Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or ...difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.
CDK11 is a cyclin-dependent kinase that controls proliferation by regulating transcription, RNA splicing, and the cell cycle. As its activity is increasingly associated with cancer, CDK11 is an ...attractive target for the development of small-molecule inhibitors. However, the development of CDK11 inhibitors with limited off-target effects against other CDKs poses a challenge based on the high conservation of sequence across family members. OTS964 is notable as it displays a measure of specificity for CDK11 in cells. To understand the basis for OTS964’s specificity for CDK11, we solved a 2.6 Å crystal structure of the CDK11 kinase domain bound to OTS964. Despite the absence of cyclin, CDK11 adopts an active-like conformation when bound to OTS964. We identified amino acids likely to contribute to the specificity of OTS964 for CDK11 and assessed their contribution to OTS964 binding by isothermal titration calorimetry (ITC) in vitro and by resistance to OTS964 in cells.
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•Determined the crystal structure of CDK11 bound to the kinase inhibitor OTS964•The CDK11 kinase domain adopts an active-like conformation•The G223S mutation causes OTS964 resistance by interfering with OTS964 binding
Kelso et al. report the crystal structure of the CDK11 kinase domain bound to the small-molecule inhibitor OTS964. Mutational analysis explores the contribution of specific contact residues toward OTS964 binding and specificity.
Essential Self-Advocacy and Transition Paradiz, Valerie; Kelso, Susan; Nelson, Andrew ...
Pediatrics,
04/2018, Letnik:
141, Številka:
Suppl 4
Journal Article
Recenzirano
Odprti dostop
As young adults with autism leave the shelter of the family home and educational entitlements and transition into adulthood, self-advocacy is indispensable. There have been many definitions of ...self-advocacy, and although they may vary in nuance and scope, self-advocacy generally refers to the ability to effectively communicate one's wants and needs. Wehmeyer and Palmer found that higher self-determination skills during the transition period lead to better outcomes in multiple facets of adult life, such as employment and financial independence.
FBXW7, which encodes a substrate‐specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post‐translational stability of various ...proteins involved in cellular proliferation. Here, using genome‐wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF‐FBXW7 E3 ligase. Further analysis showed that the CCNL1–CDK11 complex is critical at the G2‐M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss‐of‐function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.
Synopsis
The candidate oncogene CCNL1 is regulated by FBXW7 through ubiquitination and degradation in a Cul1‐dependent manner. FBXW7‐mutant cells depend on CCNL1 for growth, sensitizing them to OTS964, an inhibitor of CCNL1 kinase partner CDK11.
FBXW7‐mutant cells depend on CCNL1 for growth.
CCNL1 is a novel substrate of FBXW7.
CCNL1 mediates mitotic progression and reliance on this signalling axis sensitizes cells to CDK11 inhibitor OTS964.
The candidate oncogene CCNL1 is regulated by FBXW7 through ubiquitination and degradation in a Cul1‐dependent manner. FBXW7‐mutant cells depend on CCNL1 for growth, sensitizing them to OTS964, an inhibitor of CCNL1 kinase partner CDK11.
Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that control both transcription and progression through the cell cycle. CDKs are catalytically inactive as monomers and only ...become fully active upon heterodimerization with cyclin proteins and phosphorylation of their activation segment. Conversely, CDKs can be inhibited by binding to CDK inhibitor proteins and phosphorylation of the glycine-rich loop of the kinase domain. Whether or not a particular CDK is active depends on the abundance of their intermolecular regulators, which is controlled by the ubiquitin-proteasome system. Because CDKs play important roles in directing cell cycle events, dysregulation of their kinase activity is commonly associated with many cancers. For this reason, it is important to understand the biology underlying CDK regulation during the cell cycle and how CDKs can be targeted by small molecule kinase inhibitors. The work conducted in this thesis focuses on two different CDKs, namely CDK2 and CDK11. In Chapter 2, I investigated the binding between the CDK2-cyclin A complex and the F-box protein Skp2. Skp2 is the substrate recognition subunit of the multi-subunit E3 ubiquitin ligase, SCFSkp2. SCFSkp2 regulates CDK2 by ubiquitinating CDK inhibitor proteins. Aside from the domains required for E3 ubiquitin ligase function, Skp2 contains a disordered N-terminus that is known to bind CDK2-cyclin A via cyclin A. The functional significance of this interaction is unknown. I determined a crystal structure of the N-terminus of Skp2 bound to cyclin A and investigated how Skp2 binding cyclin A affects CDK2-cyclin A kinase activity. In Chapter 3, I focused on determining how the small molecule inhibitor OTS964 binds CDK11. OTS964 is the first reported CDK11 specific inhibitor; however, how OTS964 achieves its CDK11 specificity is unclear. To answer this question, I solved a crystal structure of OTS964 bound to CDK11 and investigated the basis of the selectivity of OTS964 by mutagenesis both in vitro and in cells.
FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various ...proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.
Skp2 and cyclin A are cell cycle regulators that control the activity of CDK2. Cyclin A acts as an activator and substrate recruitment factor of CDK2, while Skp2 mediates the ubiquitination and ...subsequent destruction of the CDK inhibitor protein p27. The N-terminus of Skp2 can interact directly with cyclin A but is not required for p27 ubiquitination. To gain insight into this poorly understood interaction, we have solved the 3.2 Å X-ray crystal structure of the N-terminus of Skp2 bound to cyclin A. The structure reveals a bi-partite mode of interaction with two motifs in Skp2 recognizing two discrete surfaces on cyclin A. The uncovered binding mechanism allows for a rationalization of the inhibitory effect of Skp2 on CDK2-cyclin A kinase activity towards RxL motif containing substrates and raises the possibility that other intermolecular regulators and substrates may use similar non-canonical modes of interaction for cyclin targeting.
Kelso et al. demonstrate that the Skp2 N-terminus contains two motifs that bind cyclin A but not cyclin E. One resembles the known RxL cyclin binding motif, but in the reverse direction. Binding of the Skp2 N-terminus to cyclin A blocks recruitment of CDK substrates.
Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely ...affects survival
. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.
Skp2 and cyclin A are cell-cycle regulators that control the activity of CDK2. Cyclin A acts as an activator and substrate recruitment factor of CDK2, while Skp2 mediates the ubiquitination and ...subsequent destruction of the CDK inhibitor protein p27. The N terminus of Skp2 can interact directly with cyclin A but is not required for p27 ubiquitination. To gain insight into this poorly understood interaction, we have solved the 3.2 Å X-ray crystal structure of the N terminus of Skp2 bound to cyclin A. The structure reveals a bipartite mode of interaction with two motifs in Skp2 recognizing two discrete surfaces on cyclin A. The uncovered binding mechanism allows for a rationalization of the inhibitory effect of Skp2 on CDK2-cyclin A kinase activity toward the RxL motif containing substrates and raises the possibility that other intermolecular regulators and substrates may use similar non-canonical modes of interaction for cyclin targeting.
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•The N terminus of Skp2 contains two separate cyclin A binding motifs•Skp2 binds to a surface on cyclin A known to bind “RxL” motifs•Skp2 contains a reverse RxL motif that binds cyclin A but not cyclin E•Skp2 binding to cyclin A blocks recruitment of RxL-containing CDK substrates
Kelso et al. demonstrate that the Skp2 N terminus contains two motifs that bind cyclin A but not cyclin E. One resembles the known RxL cyclin binding motif, but in the reverse direction. Binding of the Skp2 N terminus to cyclin A blocks recruitment of CDK substrates.
Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear ...due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high‐resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native‐like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation‐prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.
Measurements of quenched hydrogen–deuterium exchange by NMR, FTIR, and Congo red binding provide a rare, high‐resolution view of the inclusion body (IB) structure at the atomic level. Diverse mutations in superoxide dismutase (SOD1) adopt remarkably similar IB structures comprising an ensemble with some amyloid‐like structure together with extensive native‐like conformations.
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