scholarly journals Functional analysis of caspase cleavable proteoforms from the Drosophila GSK-3 gene shaggy

2020 ◽  
Author(s):  
Dagmara Korona ◽  
Daniel Nightingale ◽  
Bertrand Fabre ◽  
Michael Nelson ◽  
Bettina Fischer ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Genome Engineering ◽  
Glycogen Synthase ◽  
Kinase Domain ◽  
Protein Isoforms ◽  
Cell Junction ◽  
Loss Of Function ◽  
Caspase Cleavage ◽  
Terminal Domain ◽  
A Genome

AbstractThe Drosophila shaggy (sgg) gene encodes the major fly orthologue of Glycogen Synthase Kinase −3 (GSK-3), a key highly conserved kinase at the heart of many signalling pathways. The sgg locus is complex, encoding multiple protein isoforms that are expressed in distinct temporal and tissue-specific patterns across development. Its isoforms predominantly differ at the carboxy and amino termini due to the use of different transcriptional start sites and alternative splicing events that include internal and terminal exons. One interesting class of proteins isoforms is represented by the Sgg-PD class (Sgg46), three proteoforms that contain a large 582 amino acid N-terminal domain which contains recognition sites for caspase-mediated cleavage. Regulated cleavage at these sites by non-apoptotic caspases has previously been implicated in the regulation of Sgg activity in adult bristle development. Here, we take a genome engineering approach to introduce specific tags into this unique Sgg-PD exon and utilise these for localisation and protein interaction studies. We also generated new loss of function alleles and specific mutations in the caspase cleavage motifs. We find that loss of functions Sgg-PD class alleles are viable and fertile, but exhibit adult locomotor and bristle defects. Expression analysis of lines carrying tags on both sides of the caspase cleavage sites indicates that the cleavage is developmentally regulated during embryogenesis. Surprisingly, we found that in some cells, particularly embryonic hemocytes, the N-terminal domain released by caspase cleavage is retained while the polypeptide containing the conserved kinase domain is apparently lost. Transcriptomic analysis of embryos homozygous for the new caspase-insensitive allele indicates a role for Sgg-PD in the regulation of cytoskeletal and cell junction functions, which is supported by proteomics analysis using specific in locus tags to identify common and unique protein interaction partners with N- and C-terminal domains. Taken together, our work identifies new activities for the Sgg protein and uncovers unexpected roles for caspase cleavage in Sgg biology.

scholarly journals Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Navarro-Guerrero ◽  
Chwen Tay ◽  
Justin P. Whalley ◽  
Sally A. Cowley ◽  
Ben Davies ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Genome Engineering ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Loss Of Function ◽  
Lentiviral Transduction ◽  
Genome Wide ◽  
A Genome ◽  
Human Pathology ◽  
Induced Pluripotent

AbstractGenome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes—HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.

scholarly journals Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy

2019 ◽  
Author(s):  
Dagmara Korona ◽  
Daniel J. H. Nightingale ◽  
Bertrand Fabre ◽  
Michael Nelson ◽  
Bettina Fischer ◽  
...  
Keyword(s):  
Nervous System ◽  
Genome Engineering ◽  
Glycogen Synthase ◽  
Affinity Purification ◽  
Protein Isoforms ◽  
Phenotypic Analysis ◽  
Specific Expression ◽  
Adult Lifespan ◽  
Gsk 3Β ◽  
Developmental Signalling

AbstractThe Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoform classes we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β encoded by independent vertebrate genes. Our analysis suggests that different protein isoforms generated by alternative splicing perform distinct functions.

SGK1 mutations in DLBCL generate hyperstable protein neoisoforms that promote AKT independence.

Blood ◽  
2021 ◽  
Author(s):  
Jie Gao ◽  
Eirini Sidiropoulou ◽  
Ieuan Walker ◽  
Joanna Alicja Krupka ◽  
Karol Mizielinski ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
B Cell Lymphoma ◽  
Suppressor Gene ◽  
Kinase Domain ◽  
Tumour Suppressor Gene ◽  
Tumour Suppressor ◽  
Protein Isoforms ◽  
Loss Of Function ◽  
Over Expression ◽  
Frequent Finding

Serum and Glucocorticoid-regulated Kinase-1 (SGK1) is one of the most frequently mutated genes in Diffuse Large B Cell Lymphoma (DLBCL). However, little is known about its function or the consequence of its mutation. The frequent finding of truncating mutations has led to the widespread assumption that these represent loss-of-function variants and accordingly, that SGK1 must act as a tumour suppressor. Here we show that instead, the most common SGK1 mutations lead to production of aberrantly spliced mRNA neoisoforms in which translation is initiated from downstream methionines. The resulting N-terminal truncated protein isoforms show increased expression due to the exclusion of an N-terminal degradation domain. However, they retain a functional kinase domain, the over-expression of which renders cells resistant to AKT inhibition in part due to increased phosphorylation of GSK3B. These findings challenge the prevailing assumption that SGK1 is a tumour suppressor gene in DLBCL and provide the impetus to explore further the pharmacological inhibition of SGK1 as a therapeutic strategy for DLBCL.

scholarly journals Regulation of Chk1 by Its C-terminal Domain

2008 ◽  
Vol 19 (11) ◽  
pp. 4546-4553 ◽  
Author(s):  
Ana Kosoy ◽  
Matthew J. O'Connell
Keyword(s):  
Catalytic Domain ◽  
Kinase Domain ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Terminal Domain ◽  
C Terminus ◽  
The Face ◽  
Chk1 Kinase

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ∼200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.

scholarly journals Cell Fitness: More Than Push-Ups

2021 ◽  
Vol 22 (2) ◽  
pp. 518
Author(s):  
Adam James Ferrari ◽  
Ronny Drapkin ◽  
Rajan Gogna
Keyword(s):  
Cancer Progression ◽  
Hippo Pathway ◽  
Cancer Therapeutics ◽  
Protein Isoforms ◽  
Cell Junction ◽  
Cell Competition ◽  
Molecular Features ◽  
Oncogenic Pathways ◽  
Diagnostic Potential ◽  
The Impact

Cell competition (CC) is a feature that allows tumor cells to outcompete and eliminate adjacent cells that are deemed less fit. Studies of CC, first described in Drosophila melanogaster, reveal a diversity of underlying mechanisms. In this review, we will discuss three recent studies that expand our understanding of the molecular features governing CC. In particular, we will focus on a molecular fitness fingerprint, oncogenic pathways, and the importance of cell junction stability. A fitness fingerprint, mediated by flower (hFWE) protein isoforms, dictates that cells expressing the flower-win isoforms will outcompete adjacent flower-loss-expressing cells. The impact of the flower protein isoforms is seen in cancer progression and may have diagnostic potential. The yes-associated protein (YAP) and TAZ transcription factors, central mediators of the oncogenic Hippo pathway, elevate peritumoral fitness thereby protecting against tumor progression and provide a suppressive barrier. Similarly, COL17A1 is a key component in hemidesmosome stability, and its expression in epidermal stem cells contributes to fitness competition and aging characteristics. The contributions of these pathways to disease development and progression will help define how CC is hijacked to favor cancer growth. Understanding these features will also help frame the diagnostic and therapeutic possibilities that may place CC in the crosshairs of cancer therapeutics.

scholarly journals Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Wei-Jing Zhang ◽  
Miao Yang ◽  
Hua Fang
Keyword(s):  
Cell Apoptosis ◽  
Ischemia Reperfusion Injury ◽  
Glycogen Synthase ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

scholarly journals A Nonsense Variant in Hephaestin Like 1 (HEPHL1) Is Responsible for Congenital Hypotrichosis in Belted Galloway Cattle

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 643
Author(s):  
Thibaud Kuca ◽  
Brandy M. Marron ◽  
Joana G. P. Jacinto ◽  
Julia M. Paris ◽  
Christian Gerspach ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Homozygosity Mapping ◽  
Mendelian Inheritance ◽  
Large Animal Model ◽  
Large Animal ◽  
Loss Of Function ◽  
Protein Coding ◽  
Positional Candidate ◽  
Genome Wide ◽  
A Genome

Genodermatosis such as hair disorders mostly follow a monogenic mode of inheritance. Congenital hypotrichosis (HY) belong to this group of disorders and is characterized by abnormally reduced hair since birth. The purpose of this study was to characterize the clinical phenotype of a breed-specific non-syndromic form of HY in Belted Galloway cattle and to identify the causative genetic variant for this recessive disorder. An affected calf born in Switzerland presented with multiple small to large areas of alopecia on the limbs and on the dorsal part of the head, neck, and back. A genome-wide association study using Swiss and US Belted Galloway cattle encompassing 12 cases and 61 controls revealed an association signal on chromosome 29. Homozygosity mapping in a subset of cases refined the HY locus to a 1.5 Mb critical interval and subsequent Sanger sequencing of protein-coding exons of positional candidate genes revealed a stop gain variant in the HEPHL1 gene that encodes a multi-copper ferroxidase protein so-called hephaestin like 1 (c.1684A>T; p.Lys562*). A perfect concordance between the homozygous presence of this most likely pathogenic loss-of-function variant and the HY phenotype was found. Genotyping of more than 700 purebred Swiss and US Belted Galloway cattle showed the global spread of the mutation. This study provides a molecular test that will permit the avoidance of risk matings by systematic genotyping of relevant breeding animals. This rare recessive HEPHL1-related form of hypotrichosis provides a novel large animal model for similar human conditions. The results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 002230-9913).

scholarly journals A genome-scale protein interaction profile of Drosophila p53 uncovers additional nodes of the human p53 network

2010 ◽  
Vol 107 (14) ◽  
pp. 6322-6327 ◽  
Author(s):  
A. Lunardi ◽  
G. Di Minin ◽  
P. Provero ◽  
M. Dal Ferro ◽  
M. Carotti ◽  
...  
Keyword(s):  
Protein Interaction ◽  
A Genome ◽  
Genome Scale ◽  
P53 Network

Pyk2 and FAK differentially regulate progression of the cell cycle

2000 ◽  
Vol 113 (17) ◽  
pp. 3063-3072 ◽  
Author(s):  
J. Zhao ◽  
C. Zheng ◽  
J. Guan
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Expression System ◽  
Chimeric Protein ◽  
Kinase Domain ◽  
Cycle Progression ◽  
Erk Activation ◽  
Terminal Domain ◽  
Cycle Regulation

We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G(1) to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant (Δ)C14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.

The domino gene of Drosophila encodes novel members of the SWI2/SNF2 family of DNA-dependent ATPases, which contribute to the silencing of homeotic genes

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1429-1441 ◽  
Author(s):  
M.L. Ruhf ◽  
A. Braun ◽  
O. Papoulas ◽  
J.W. Tamkun ◽  
N. Randsholt ◽  
...  
Keyword(s):  
Polytene Chromosomes ◽  
Gene Mutations ◽  
Polycomb Group ◽  
Protein Isoforms ◽  
Homeotic Genes ◽  
Loss Of Function ◽  
Trithorax Group ◽  
A Subunit ◽  
The Common ◽  
Hematopoietic Disorders

The Drosophila domino gene has been isolated in a screen for mutations that cause hematopoietic disorders. Generation and analysis of loss-of-function domino alleles show that the phenotypes are typical for proliferation gene mutations. Clonal analysis demonstrates that domino is necessary for cell viability and proliferation, as well as for oogenesis. domino encodes two protein isoforms of 3202 and 2498 amino acids, which contain a common N-terminal region but divergent C termini. The common region includes a 500 amino acid DNA-dependent ATPase domain of the SWI2/SNF2 family of proteins, which function via interaction with chromatin. We show that, although domino alleles do not exhibit homeotic phenotypes by themselves, domino mutations enhance Polycomb group mutations and counteract Trithorax group effects. The Domino proteins are present in large complexes in embryo extracts, and one isoform binds to a number of discrete sites on larval polytene chromosomes. Altogether, the data lead us to propose that domino acts as a repressor by interfering with chromatin structure. This activity is likely to be performed as a subunit of a chromatin-remodeling complex.

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