Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence.

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing

Blueprint of the Intramolecular Regulatory Mechanism of Eukaryotic Protein Kinases

Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through a highly conserved catalytic domain. Previous computational studies proposed a model stating that a properly assembled non-linear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site- directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed 4 unique structural conformations of the R- spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors. The catalytic core of EPKs oscillates between inactive and active states as well as toggling between open and closed conformations when active. Currently, the intramolecular interactions that regulate this dynamic behavior are not well understood. Here, we show that there are at least two possible mechanisms regulating this dynamics. The first mechanism involves the highly conserved salt bridge between a lysine from the [Beta]3- strand and a glutamate from the [alpha]C-helix as well as a hydrogen bond that only forms when the activation loop is phosphorylated. The second mechanism involves an ensemble of hydrophobic interactions within the nonlinear motifs known as the Regulatory spine and Shell. Our findings also show that the highly conserved [Beta]3- lysine serves as a "catalytic synchronization hub" that aligns and positions the dynamic components required for catalysi

Blueprint of the Intramolecular Regulatory Mechanism of Eukaryotic Protein Kinases

Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through a highly conserved catalytic domain. Previous computational studies proposed a model stating that a properly assembled non-linear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site- directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed 4 unique structural conformations of the R- spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors. The catalytic core of EPKs oscillates between inactive and active states as well as toggling between open and closed conformations when active. Currently, the intramolecular interactions that regulate this dynamic behavior are not well understood. Here, we show that there are at least two possible mechanisms regulating this dynamics. The first mechanism involves the highly conserved salt bridge between a lysine from the [Beta]3- strand and a glutamate from the [alpha]C-helix as well as a hydrogen bond that only forms when the activation loop is phosphorylated. The second mechanism involves an ensemble of hydrophobic interactions within the nonlinear motifs known as the Regulatory spine and Shell. Our findings also show that the highly conserved [Beta]3- lysine serves as a "catalytic synchronization hub" that aligns and positions the dynamic components required for catalysi

The History of the Decline and Fall of News of the World : How Legitimacy Upholds the License to Operate

This thesis provides a biographical case study of the crisis faced by News of the World, after revelations of unethical journalistic practices within the newspaper. A scandal that rapidly moved up the organizational hierarchy and spread globally, affecting stakeholders amongst all its networks from local British communities to its parent company News Corporation and its main owners, the Murdoch family. Corporate social responsibility and stakeholder management is presented as a key factor in order to uphold and obtain the society’s permission to operate. The study shows the importance of being a good corporate citizen, not solely as goodwill but in order to gain a parachute that can moderate the fall when facing a crisis

From neurodevelopment to neurodegeneration: utilizing human stem cell models to gain insight into Down syndrome.

Down syndrome (DS), caused by triplication of chromosome 21, is the most frequent aneuploidy observed in the human population and represents the most common genetic form of intellectual disability and early-onset Alzheimer's disease (AD). Individuals with DS exhibit a wide spectrum of clinical presentation, with a number of organs implicated including the neurological, immune, musculoskeletal, cardiac, and gastrointestinal systems. Decades of DS research have illuminated our understanding of the disorder, however many of the features that limit quality of life and independence of individuals with DS, including intellectual disability and early-onset dementia, remain poorly understood. This lack of knowledge of the cellular and molecular mechanisms leading to neurological features of DS has caused significant roadblocks in developing effective therapeutic strategies to improve quality of life for individuals with DS. Recent technological advances in human stem cell culture methods, genome editing approaches, and single-cell transcriptomics have provided paradigm-shifting insights into complex neurological diseases such as DS. Here, we review novel neurological disease modeling approaches, how they have been used to study DS, and what questions might be addressed in the future using these innovative tools

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation

Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers

Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase