scholarly journals Structural and Functional Determinants of Conserved Lipid Interaction Domains of Inward Rectifying Kir6.2 Channels

2002 ◽  
Vol 119 (6) ◽  
pp. 581-591 ◽  
Author(s):  
Catherine A. Cukras ◽  
Iana Jeliazkova ◽  
Colin G. Nichols
Keyword(s):  
Critical Role ◽  
Katp Channels ◽  
Pleckstrin Homology Domain ◽  
Membrane Association ◽  
Kir Channel ◽  
M1 Receptor ◽  
Interaction Domains ◽  
Mechanistic Basis ◽  
Α Helix

All members of the inward rectifiier K+ (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of KATP channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and KATP channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C δ1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor–mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple β-strands and a conserved α-helix (residues ∼306–311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a α-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

Cationicity and hydrophobicity enhance the cytotoxic potency of Phoratoxin C anticancer peptide analogues against triple negative breast cancer cells

2021 ◽  
Vol 17 ◽  
Author(s):  
Chu Xin Ng ◽  
Cheng Foh Le ◽  
Sau Har Lee
Keyword(s):  
Cancer Cells ◽  
Anticancer Agents ◽  
Breast Cancer Cells ◽  
Critical Role ◽  
Three Dimensional ◽  
Vero Cells ◽  
Anticancer Peptides ◽  
Α Helix ◽  
Peptide Analogues

Background: Anticancer peptides (ACPs) have received increasing attention as a promising class of novel anticancer agents owing to its potent and rapid cytotoxic properties. In this study, we aim to investigate the effects of cationicity and hydrophobicity in modulating the cytotoxicity of PtxC, a class of ACP from the leafy mistletoe Phoradendron tomentosum against the MDA-MB-231 and Vero cells. Method: We designed a series of four PtxC analogues (PA1 – PA4) by residual substitutions with specific amino acids to introduce the specific charge and hydrophobicity alterations to the analogues. The cytotoxicity strength of the PtxC analogues on MDA-MB-231 and Vero cells were tested by using MTT assay at 24 hours post treatment. Results: PA1, PA2 and PA4 displayed marked increases in cytotoxicity against both MDA-MB-231 and Vero cells and can be ranked in the order of PA2 > PA4 > PA1 > PtxC > PA3. Sequence-activity relationship analyses of the designed analogues showed that an increase in the level of cationicity and hydrophobicity correlated well with the enhanced cytotoxic activity of PtxC analogues. This was observed with PA1 (netC +8) and PA2 (netC +10) in comparison to PtxC (netC +7). Similar finding was observed for PA4 (GRAVY +0.070) in contrast to PtxC (GRAVY -0.339). Three-dimensional modelling predicted a double α-helix structure in PtxC class of ACP. The larger first helix in PA2 and PA4 was suggested to be responsible for the enhanced cytotoxicity observed. Conclusion: The critical role of cationicity and hydrophobicity in enhancing cytotoxicity of PtxC class of ACPs were clearly demonstrated in our study. The current findings could be extrapolated to benefit peptide design strategy in other classes of ACPs toward the discovery of highly potent ACPs against cancer cells as potential novel therapeutic agents.

scholarly journals Critical Role of the Pleckstrin Homology Domain in Dbs Signaling and Growth Regulation

2003 ◽  
Vol 278 (23) ◽  
pp. 21188-21196 ◽  
Author(s):  
Ernesto J. Fuentes ◽  
Antoine E. Karnoub ◽  
Michelle A. Booden ◽  
Channing J. Der ◽  
Sharon L. Campbell
Keyword(s):  
Growth Regulation ◽  
Critical Role ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

Factor XII Contact Activation

2017 ◽  
Vol 43 (08) ◽  
pp. 814-826 ◽  
Author(s):  
Clément Naudin ◽  
Elena Burillo ◽  
Stefan Blankenberg ◽  
Lynn Butler ◽  
Thomas Renné
Keyword(s):  
Critical Role ◽  
Factor Xii ◽  
Contact Activation ◽  
Kinin System ◽  
Disease States ◽  
Charged Surfaces ◽  
Mechanistic Basis ◽  
Kallikrein Kinin System

AbstractContact activation is the surface-induced conversion of factor XII (FXII) zymogen to the serine protease FXIIa. Blood-circulating FXII binds to negatively charged surfaces and this contact to surfaces triggers a conformational change in the zymogen inducing autoactivation. Several surfaces that have the capacity for initiating FXII contact activation have been identified, including misfolded protein aggregates, collagen, nucleic acids, and platelet and microbial polyphosphate. Activated FXII initiates the proinflammatory kallikrein-kinin system and the intrinsic coagulation pathway, leading to formation of bradykinin and thrombin, respectively. FXII contact activation is well characterized in vitro and provides the mechanistic basis for the diagnostic clotting assay, activated partial thromboplastin time. However, only in the past decade has the critical role of FXII contact activation in pathological thrombosis been appreciated. While defective FXII contact activation provides thromboprotection, excess activation underlies the swelling disorder hereditary angioedema type III. This review provides an overview of the molecular basis of FXII contact activation and FXII contact activation–associated disease states.

scholarly journals VCP/p97 controls signals of the ERK1/2 pathway transmitted via the Shoc2 scaffolding complex: novel insights into IBMPFD pathology

2019 ◽  
Vol 30 (14) ◽  
pp. 1655-1663 ◽  
Author(s):  
HyeIn Jang ◽  
Eun Ryoung Jang ◽  
Patricia G. Wilson ◽  
Daniel Anderson ◽  
Emilia Galperin
Keyword(s):  
Atpase Activity ◽  
Frontotemporal Dementia ◽  
Inclusion Body ◽  
Critical Role ◽  
Germline Mutations ◽  
Aaa Atpase ◽  
Ubiquitin System ◽  
Signaling Axis ◽  
Mechanistic Basis

Valosin-containing protein (VCP), also named p97, is an essential hexameric AAA+ ATPase with diverse functions in the ubiquitin system. Here we demonstrate that VCP is critical in controlling signals transmitted via the essential Shoc2-ERK1/2 signaling axis. The ATPase activity of VCP modulates the stoichiometry of HUWE1 in the Shoc2 complex as well as HUWE1-mediated allosteric ubiquitination of the Shoc2 scaffold and the RAF-1 kinase. Abrogated ATPase activity leads to augmented ubiquitination of Shoc2/RAF-1 and altered phosphorylation of RAF-1. We found that in fibroblasts from patients with inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia (IBMPFD) that harbor germline mutations in VCP, the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced. This study provides a mechanistic basis for the critical role of VCP in the regulation of the ERK1/2 pathway and reveals a previously unrecognized function of the ERK1/2 pathway in the pathogenesis of IBMPFD.

Photoprotection in chilling-sensitive and -resistant plants illuminated at a chilling temperature: role of the xanthophyll cycle in the protection against lumen acidification

2000 ◽  
Vol 27 (7) ◽  
pp. 669 ◽  
Author(s):  
Chang-Cheng Xu ◽  
Liangbi Li ◽  
Tingyun Kuang
Keyword(s):  
Xanthophyll Cycle ◽  
Higher Plants ◽  
Critical Role ◽  
Reaction Centers ◽  
The Other ◽  
Strong Positive Correlation ◽  
Chilling Temperature ◽  
Chilling Sensitivity ◽  
Mechanistic Basis

The role of the xanthophyll cycle in the protection against photoinhibition of photosystem II (PSII) induced by chilling in moderate light was investigated in leaves of eight species or varieties of higher plants differing widely in chilling sensitivity. The extent of photoinhibition measured as the increase in the slowly reversible fluorescence quenching (qI) was found not to correlate with the overall amount of zeaxanthin formed during photo-inhibitory treatment. On the other hand, a strong, positive correlation existed between qI and the rate difference between the development of the rapidly relaxing, DpH-dependent quenching component (qf) and the formation of zeaxanthin (DR) across all examined species or varieties. There was also found to be a weaker, negative correlation between qI and the rate of zeaxanthin formation. Ascorbate feeding markedly increased the resistance to chilling-induced photoinhibition mainly by increasing the rate of zeaxanthin formation and therefore by decreasing DR. The possible implications of the present findings in explaining the mechanistic basis for the reversible, sustained photo-inhibition are discussed. It is suggested that the xanthophyll cycle may play a critical role in the protection of the thylakoid lumen against over-acidification and the resulting photoinhibition of PSII reaction centers.

ANALYSIS OF HUMAN IL-2/IL-2 RECEPTOR β CHAIN INTERACTIONS: MONOCLONAL ANTIBODY H2-8 AND NEW IL-2 MUTANTS DEFINE THE CRITICAL ROLE OF α HELIX-A OF IL-2

Cytokine ◽  
1997 ◽  
Vol 9 (7) ◽  
pp. 488-498 ◽  
Author(s):  
Ralph Eckenberg ◽  
Di Xu ◽  
Jean-Louis Moreau ◽  
Marc Bossus ◽  
Jean-Claude Mazié ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Critical Role ◽  
Α Helix ◽  
Β Chain

scholarly journals Bioactive lipid: A novel diagnostic approach for retinoblastoma in clinical management

2021 ◽  
Vol 0 ◽  
pp. 1-4
Author(s):  
Ankit Srivastava ◽  
Bimal Prasad Jit ◽  
Rutumbara Dash ◽  
Manasa Kumar Panda
Keyword(s):  
Lysophosphatidic Acid ◽  
Lipid A ◽  
Critical Role ◽  
Bioactive Lipids ◽  
Cellular Functions ◽  
Epidemiological Evidence ◽  
Diagnostic Biomarkers ◽  
Sphingosine 1 Phosphate ◽  
Mechanistic Basis

Bioactive lipids, presumably lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), play a critical role in regulating an array of cellular functions ranging from cellular fate determination, inflammation, immunity, and cancer. Epidemiological evidence suggests that both the metabolites play a prominent role in the development and progression of oncogenic phenotype in a variety of cancers including breast, colorectal, pancreatic, and lymphoma. Previous studies have demonstrated the possible association of LPA, S1P and their receptor in regulating the pathogenesis of retinoblastoma, however, the exact mechanism involved in this event has not been studied in detail. Importantly, understating the mechanistic basis of LPA and S1P regulation is of utmost significance, as far the phenotypical complexity of retinoblastoma (RB) is concerned. Findings from the recent investigations elucidate the prospective role of S1P in provoking the chemoresistant behavior of RB cells for etoposide. In this context, the current paper will enable the identification of novel diagnostic biomarkers and therapeutic targets for better treatment and clinical efficacy in children with RB.

scholarly journals Flexible pivoting of dynamin PH-domain catalyzes fission: Insights into molecular degrees of freedom

2019 ◽  
Author(s):  
K. K. Baratam ◽  
K. Jha ◽  
A. Srivastava
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Synaptic Vesicle ◽  
Molecular Level ◽  
Pleckstrin Homology Domain ◽  
Membrane Association ◽  
Membrane Interactions ◽  
Vesicle Recycling ◽  
Membrane Fission

ABSTRACTThe neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) with its centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed, albeit at much slower rates, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, even when the PHD is present, the length of the dynamin scaffold and in turn the membrane remodeling and fission rates are severely restricted with mutations such as I533A on membrane-interacting variable loop 1 (VL1) of PHD. These observations suggest that PIP2-containing membrane interactions of PHD could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling methods (metadynamics and umbrella sampling) to explore PHD-membrane interactions. Our results reveal that: (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, (b) that PHD engages another loop (VL4) for membrane association, which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane – a mechanism we believe may be important for high fidelity dynamin collar assembly on the membrane. (c) Through analyses of our trajectories data and free-energy calculations on membrane-bound WT and mutant systems, we also identify key residues on multiple VLs that stabilizes PHD membrane association. And we suggest experiments to explore the ability of PHD to associate with membrane in orientations that favors faster fission. Together, these insights provide a molecular-level understanding of the “catalytic” role of the PHD in dynamin-mediated membrane fission.SIGNIFICANCEDynamin, a large multi-domain GTPase, remodels the membrane by self-assembling onto the neck of a budding vesicle and induces fission by its energy driven conformational changes. In this work, we use multi-scale molecular simulations to probe the role of dynamin’s pleckstrin-homology domain (PHD), which facilitates membrane interactions. Notably, PHD is dispensable for fission as is the case with extant bacterial and mitochondrial dynamins. However, reconstitution experiments suggest that the functional role of PHD in neuronal-membrane goes beyond that of an adaptor domain as it possibly ‘expedites’ the fission reaction during synaptic vesicle recycling. We provide a molecular-dynamics picture of how PHDs make membranes more pliable for fission and suggest new insights into the molecular-level processes driving the expedited fission behavior.

scholarly journals The Role of NH2-terminal Positive Charges in the Activity of Inward Rectifier KATP Channels

2002 ◽  
Vol 120 (3) ◽  
pp. 437-446 ◽  
Author(s):  
C.A. Cukras ◽  
I. Jeliazkova ◽  
C.G. Nichols
Keyword(s):  
Katp Channels ◽  
Inward Rectifier ◽  
Structural Determinants ◽  
Open Probability ◽  
Channel Function ◽  
Kir Channel ◽  
Membrane Spanning ◽  
Relationship Of ◽  
The Relationship

Approximately half of the NH2 terminus of inward rectifier (Kir) channels can be deleted without significant change in channel function, but activity is lost when more than ∼30 conserved residues before the first membrane spanning domain (M1) are removed. Systematic replacement of the positive charges in the NH2 terminus of Kir6.2 with alanine reveals several residues that affect channel function when neutralized. Certain mutations (R4A, R5A, R16A, R27A, R39A, K47A, R50A, R54A, K67A) change open probability, whereas an overlapping set of mutants (R16A, R27A, K39A, K47A, R50A, R54A, K67A) change ATP sensitivity. Further analysis of the latter set differentiates mutations that alter ATP sensitivity as a consequence of altered open state stability (R16A, K39A, K67A) from those that may affect ATP binding directly (K47A, R50A, R54A). The data help to define the structural determinants of Kir channel function, and suggest possible structural motifs within the NH2 terminus, as well as the relationship of the NH2 terminus with the extended cytoplasmic COOH terminus of the channel.

scholarly journals Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis

2021 ◽  
Vol 22 (19) ◽  
pp. 10862
Author(s):  
Carolina F. Rodrigues ◽  
Patrícia T. Borges ◽  
Magali F. Scocozza ◽  
Diogo Silva ◽  
André Taborda ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Critical Role ◽  
Wild Type ◽  
Positive Functional ◽  
Heme Pocket ◽  
Active Site Cavity ◽  
Loop Regions ◽  
Α Helix

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme’s overall stability by 2 kcal mol−1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

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