ORCID Profile
0000-0001-7156-9403
Current Organisations
Wayne State University School of Medicine
,
University of Leeds
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Publisher: Springer Science and Business Media LLC
Date: 05-2020
DOI: 10.1038/S41467-020-15702-1
Abstract: The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA s les an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Elsevier BV
Date: 07-2018
Publisher: Cold Spring Harbor Laboratory
Date: 04-08-2023
DOI: 10.1101/2023.08.01.23292554
Abstract: Generalised Lymphatic Dysplasia (GLD) is characterised by widespread lymphoedema, with at least one of the following: fetal hydrops, intestinal or pulmonary lymphangiectasia, pleural effusions, pericardial effusions and ascites. Satisfactory medical therapies are lacking. A genetic association has been identified that prevents expression or surface trafficking of PIEZO1, a subunit of mechanically activated calcium-permeable channels. However, PIEZO1 is a large and highly polymorphic gene and interpretation of variants identified in this gene can be challenging. PIEZO1- related GLD with non-immune fetal hydrops is autosomal recessive, however, heterozygous variants in PIEZO1 (often gain-of-function) causing Dehydrated Hereditary Stomatocytosis (DHS) (a relative mild anaemia), may also present with perinatal non-immune hydrops (not caused by anaemia). Here we sought to develop methods to confirm pathogenicity of missense variants of uncertain significance in PIEZO1 , to gain deeper understanding and pharmacological solutions. Four novel GLD-associated missense variants in PIEZO1 are identified that express and surface localise as full-length protein but with reduced or abolished mechanically activated channel function. Yoda1, a small-molecule agonist, functionally rescues the channels and their physiological regulation by mechanical force and hypo-osmolality. The GLD-associated variants mediate intracellular calcium release as well as calcium entry, suggesting two pools of channels and opportunity for increased rescue through access to the intracellular pool. New Yoda1 analogues are also identified that improve rescue. The functional assays have assisted the interpretation of the variants of uncertain significance as the data suggest loss of PIEZO1 force sensing as a cause of the GLD observed in the patients. The potential to pharmacologically overcome the loss of force sensing was demonstrated and supports the concept of stimulation of PIEZO1 with an agonist to address wide-ranging problems of lymphatic insufficiency. Previously unrecognised variants in PIEZO1 that associate with GLD are identified and characterised and pathogenicity confirmed The variants encode single amino acid changes that inhibit PIEZO1 channel activation by physiological mechanical forces A small-molecule agonist rescues the channels and their physiological regulation Variants are partly intracellular, suggesting an opportunity for improved rescue through the use of intracellular-acting agonists New agonists are identified that improve rescue, suggesting routes to medical therapies for GLD and potentially other disorders of lymphatic insufficiency
Publisher: Public Library of Science (PLoS)
Date: 30-03-2015
Publisher: Springer Science and Business Media LLC
Date: 14-12-2020
DOI: 10.1038/S42003-020-01419-W
Abstract: The β-barrel assembly machinery (BAM) catalyses the folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The β-barrel domain of BamA is in a ‘lateral open’ conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM’s lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid ‘disruptase’ activity of BAM, suggested to be an important part of its functional mechanism.
Publisher: Walter de Gruyter GmbH
Date: 09-2017
Abstract: The aim of this study was to determine the association between chronic placental inflammation and amniotic fluid (AF) markers of maternal anti-fetal rejection as well as the presence of microorganisms in the AF fluid of patients with fetal death. This cohort study included 40 patients with fetal death whose placentas were examined for chronic inflammatory lesions and whose AF chemokine ligand (CXCL)10 and interleukin (IL)-6 concentrations were determined by immunoassays. AF was processed for bacteria, mycoplasmas and viruses using cultivation and molecular microbiologic techniques (i.e. PCR-ESI/MS). (1) The most prevalent placental findings were maternal vascular underperfusion (63.2%, 24/38), followed by chronic inflammatory lesions (57.9%, 22/38) (2) chronic chorioamnionitis (18/38) was three times more frequent than villitis of unknown etiology (6/38) (3) an elevated AF CXCL10 concentration (above the 95 th centile) was present in 60% of the cases, and a receiver operating characteristics (ROC)-derived cut-off of 2.9 ng/mL had a sensitivity of 73% and a specificity of 75% in the identification of chronic placental inflammatory lesions (4) only five cases had microbial invasion of the amniotic cavity, and the presence of microorganisms did not correlate with chronic placental inflammation. In women with unexplained fetal death, there is an association between elevated AF CXCL10 and chronic placental inflammatory lesions. Therefore, we conclude that a subset of patients with fetal death may have endured a breakdown of maternal-fetal tolerance, which cannot be attributed to microorganisms in the amniotic cavity.
Publisher: Walter de Gruyter GmbH
Date: 2015
Abstract: Recent studies indicate that clinical chorioamnionitis is a heterogeneous condition and only approximately one-half of the patients have bacteria in the amniotic cavity, which is often associated with intra-amniotic inflammation. The objective of this study is to characterize the nature of the inflammatory response within the amniotic cavity in patients with clinical chorioamnionitis at term according to the presence or absence of 1) bacteria in the amniotic cavity and 2) intra-amniotic inflammation. A retrospective cross-sectional case-control study was conducted to examine cytokine and chemokine concentrations in the amniotic fluid (AF). Cases consisted of women with clinical chorioamnionitis at term (n=45). Controls were women with uncomplicated pregnancies at term who did not have intra-amniotic inflammation and were in labor (n=24). Women with clinical chorioamnionitis were classified according to the results of AF cultures, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and AF concentration of interleukin-6 (IL-6) into those: 1) without intra-amniotic inflammation, 2) with microbial-associated intra-amniotic inflammation, and 3) with intra-amniotic inflammation without detectable bacteria. The AF concentrations of 29 cytokines/chemokines were determined using sensitive and specific V-PLEX immunoassays. 1) The AF concentrations of pro- and anti-inflammatory cytokines/chemokines such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), macrophage inflammatory protein-1 beta (MIP-1β), and interleukin-8 (IL-8) (except Eotaxin-3) were significantly higher in women with clinical chorioamnionitis at term than in controls (term labor without intra-amniotic inflammation) 2) patients with microbial-associated intra-amniotic inflammation, and those with intra-amniotic inflammation without detectable bacteria, had a dramatic differential expression of cytokines and chemokines in AF compared to patients with spontaneous labor without intra-amniotic inflammation. However, no difference could be detected in the pattern of the intra-amniotic inflammatory response between patients with intra-amniotic inflammation with and without detectable bacteria and 3) in patients with clinical chorioamnionitis at term but without intra-amniotic inflammation, the behavior of cytokines and chemokines in the AF was similar to those in spontaneous labor at term. Patients with clinical chorioamnionitis who had microbial-associated intra-amniotic inflammation or intra-amniotic inflammation without detectable bacteria had a dramatic upregulation of the intra-amniotic inflammatory response assessed by amniotic fluid concentrations of cytokines. A subset of patients with term clinical chorioamnionitis does not have intra-amniotic infection/inflammation, as demonstrated by elevated AF concentrations of inflammation-related proteins, when compared to women in term labor with uncomplicated pregnancies, suggesting over-diagnosis. These observations constitute the first characterization of the cytokine/chemokine network in the amniotic cavity of patients with clinical chorioamnionitis at term.
Publisher: Public Library of Science (PLoS)
Date: 26-07-2016
Publisher: Cold Spring Harbor Laboratory
Date: 04-2021
DOI: 10.1101/2021.04.01.438050
Abstract: The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily s le the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL s les a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.
Location: United States of America
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Antreas Kalli.