Biology subjects

Lane, D.

Publications and source records attributed to Lane, D..

5 recordsLinked to original sources

bioRxiv2023

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood so far. To date there have been no reports of the response of the pneumococcal competence regulon to changes in the physical conditions of the environment, oxidative potential and temperature. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that certain other competence induction stimuli are not conveyed by the HtrA-dependent pathway. This finding strongly supports that competence can be induced by any of several independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus. Authors summaryStreptococcus pneumoniae is a commensal bacterium and an opportunistic pathogen of humans. Certain environmental stimuli, such as a variety of antibiotics targeting distinct cellular functions, trigger the induction of the distinct physiological state of competence, in which cells can, among other things, import and integrate external DNA. Competence is thus considered a general stress response in this highly adaptable species. To understand the role of competence in pneumococcal interaction with its host and in pathogenicity, we attempted to decipher the pathways that enable appropriate reactions to environmental stress, and have focused here on induction of competence by elevation of temperature to levels similar to that of a host in fever. We found that elevated temperature raises the basal expression level of the competence control operon, and thus lowers the threshold of transition to full competence induction. By genetic characterisation of the thermal induction pathway of competence, we demonstrated that the chaperone/protease HtrA is essential to relaying of the thermal signal but is not involved in transmitting other stimuli such as those arising from the presence of certain antibiotics. Our work supports the view that competence can be induced through various pathways in response to distinct aggressions, but also by fever-like bursts of temperature that the pneumococcus could face in its natural habitat.

microbiology↗

bioRxiv2020

Both DNA binding domains of p53 are required for its ultra rapid recruitment to sites of UV damage

p53 concentrates at DNA damage sites within two seconds upon UV laser micro-irradiation. Structural analysis shows that this very rapid response requires both the DNA binding and C-terminal domains of p53. This early recruitment response is also PARP-dependent. As mutations within the DNA binding domain of p53, that are commonly associated with cancer also inhibit this rapid binding, we suggest that this is an important initial step for p53 function as a tumor suppressor. One Sentence Summaryp53 is an early responder to DNA damage

cell biology↗

bioRxiv2019

Role of centromere sites in activation of ParB proteins for partition complex assembly

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. We conclude that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

microbiology↗

bioRxiv2019

Macrocyclization of an all-D linear peptide improves target affinity and imparts cellular activity: A novel stapled α-helical peptide modality

Peptide-based inhibitors hold great potential for targeted modulation of intracellular protein-protein interactions (PPIs) by leveraging vast chemical space relative to primary structure via sequence diversity as well as conformationally through varying secondary and tertiary structures. However, the development of peptide therapeutics has been hindered because of their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies address these issues to varying degrees. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may address these issues by i) restricting conformational freedom to improve target affinities, ii) improving proteolytic resistance, and iii) enhancing cell permeability. Conversely, molecules constructed entirely from D-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-D -helical stapled and stitched peptides. As a template, we used a recently reported all D-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-D-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all D-peptide was determined to exhibit increased -helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

biochemistry↗

bioRxiv2018

Endogenous protection from ischemic brain injury by preconditioned monocytes

Exposure to low dose lipopolysaccharide prior to cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning. While it is well established that lipopolysaccharide-preconditioning induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by preconditioning and we tested whether monocytes may be reprogrammed by ex vivo lipopolysaccharide exposure thus modulating the inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose lipopolysaccharide induces a distinct subclass of CD115+Ly6Chi monocytes that protect the brain after transient middle cerebral artery occlusion in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 hours after transient middle cerebral artery occlusion reduced brain injury. Gene expression and functional studies showed that IL-10, iNOS and CCR2 in monocytes are essential for the neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to lipopolysaccharide and then injected into male mice after stroke. Cell tracking studies showed that protective monocytes are mobilized from the spleen and reach brain and meninges, wherein they suppressed post-ischemic inflammation and neutrophils influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable to protect the brain with an extended therapeutic window, and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes into patients with ischemic stroke.\n\nSignificance StatementInflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using LPS preconditioning as an approach to induce ischemic tolerance in mice, we found the generation of neuroprotective monocytes within the spleen from where they traffic to the brain and meninges suppressing post-ischemic inflammation. Importantly, systemic LPS preconditioning can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of bone marrow mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

neuroscience↗