scholarly journals Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors

1998 ◽  
Vol 331 (2) ◽  
pp. 375-379 ◽  
Author(s):  
Irene YIALLOUROS ◽  
Stamatia VASSILIOU ◽  
Athanasios YIOTAKIS ◽  
Robert ZWILLING ◽  
Walter STÖCKER ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Critical Role ◽  
Fold Increase ◽  
Rational Basis ◽  
Inhibitor Binding ◽  
Parent Molecule ◽  
Transition State Analogue ◽  
Morphogenetic Protein ◽  
Binding Behaviour

A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1·s-1) and dissociation (koff = 4.1×10-6 s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-O-methyl, Ki = 14 µM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stöcker (1996) Nature Struct. Biol. 3, 671–675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique ‘tyrosine switch ’ in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.

scholarly journals The Potential of Nail Mini-Organ Stem Cells in Skin, Nail and Digit Tips Regeneration

2021 ◽  
Vol 22 (6) ◽  
pp. 2864
Author(s):  
Anna Pulawska-Czub ◽  
Tomasz D. Pieczonka ◽  
Paula Mazurek ◽  
Krzysztof Kobielak
Keyword(s):  
Stem Cells ◽  
Critical Role ◽  
Nail Plate ◽  
Molecular Characteristics ◽  
Continuous Growth ◽  
Physiological Conditions ◽  
Morphogenetic Protein ◽  
Slow Cycling ◽  
Bifunctional Properties

Nails are highly keratinized skin appendages that exhibit continuous growth under physiological conditions and full regeneration upon removal. These mini-organs are maintained by two autonomous populations of skin stem cells. The fast-cycling, highly proliferative stem cells of the nail matrix (nail stem cells (NSCs)) predominantly replenish the nail plate. Furthermore, the slow-cycling population of the nail proximal fold (nail proximal fold stem cells (NPFSCs)) displays bifunctional properties by contributing to the peri-nail epidermis under the normal homeostasis and the nail structure upon injury. Here, we discuss nail mini-organ stem cells’ location and their role in skin and nail homeostasis and regeneration, emphasizing their importance to orchestrate the whole digit tip regeneration. Such endogenous regeneration capabilities are observed in rodents and primates. However, they are limited to the region adjacent to the nail’s proximal area, indicating the crucial role of nail mini-organ stem cells in digit restoration. Further, we explore the molecular characteristics of nail mini-organ stem cells and the critical role of the bone morphogenetic protein (BMP) and Wnt signaling pathways in homeostatic nail growth and digit restoration. Finally, we investigate the latest accomplishments in stimulating regenerative responses in regeneration-incompetent injuries. These pioneer results might open up new opportunities to overcome amputated mammalian digits and limbs’ regenerative failures in the future.

scholarly journals Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

2020 ◽  
Author(s):  
Carmen Lopez ◽  
Mingfeng Cao ◽  
Zhanyi Yao ◽  
Zengyi Shao
Keyword(s):  
Yarrowia Lipolytica ◽  
Plasmid Stability ◽  
Critical Role ◽  
Fold Increase ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform ◽  
Regulatory Dna ◽  
Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

scholarly journals The role of endothelial PI3Kγ activity in neutrophil trafficking

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 150-157 ◽  
Author(s):  
Kamal D. Puri ◽  
Teresa A. Doggett ◽  
Ching-Yu Huang ◽  
Jason Douangpanya ◽  
Joel S. Hayflick ◽  
...  
Keyword(s):  
Critical Role ◽  
Fold Increase ◽  
Necrosis Factor Alpha ◽  
Catalytic Subunits ◽  
Injury Model ◽  
Lung Injury Model ◽  
Factor Alpha ◽  
Phosphoinositide 3 Kinase

Phosphoinositide 3-kinase gamma (PI3Kγ) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kγ activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110γ–/– mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110γ–/– microvessels in vivo in response to tumor necrosis factor alpha (TNFα). This alteration in adhesion was further augmented by a deficiency in p110δ, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin–mediated adhesion in p110γ–/– mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-κB)–induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.

scholarly journals Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells

2014 ◽  
Vol 307 (1) ◽  
pp. G66-G76 ◽  
Author(s):  
Hae-Ki Min ◽  
Silvia Sookoian ◽  
Carlos J. Pirola ◽  
Jianfeng Cheng ◽  
Faridoddin Mirshahi ◽  
...  
Keyword(s):  
Liver Disease ◽  
Metabolic Profiling ◽  
Critical Role ◽  
Fold Increase ◽  
Liver Metabolism ◽  
Hepatoma Cells ◽  
Triacylglycerol Lipase ◽  
Nonalcoholic Fatty Liver ◽  
Triglyceride Accumulation

PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling.

Differential Effects of Protein Disulfide Isomerase (PDI) Inhibitors On the Expression of Tissue Factor Procoagulant Activity (TF PCA) by AML Blasts

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1112-1112
Author(s):  
Cornelia Fischer ◽  
Brigitte Spath ◽  
Ali Amirkhosravi ◽  
Walter Fiedler ◽  
Carsten Bokemeyer ◽  
...  
Keyword(s):  
Critical Role ◽  
Leukemic Cell ◽  
Fold Increase ◽  
Myelogenous Leukemia ◽  
Short Term ◽  
Short Term Treatment ◽  
Hl60 Cells ◽  
Short Term Exposure

Abstract Abstract 1112 Acute myelogenous leukemia (AML) may be complicated by DIC. TF plays a critical role in AML-associated coagulopathy, and induction of apoptosis significantly increases TF PCA on leukemic blasts, mainly via phosphatidylserine (PS) membrane exposure. However, PDI, a thiol isomerase with oxidoreductase and chaperone activity, has also been implicated in cellular TF regulation. Particularly, PDI inhibitors have been shown to exert antithrombotic activity in animal models. Besides its predominant localization in the endoplasmic reticulum, PDI is present on cell surfaces, where it may represent a promising therapeutic target. We investigated the effect of PDI inhibitors on the expression of TF PCA by leukemic HL60 and THP1 cells to explore their potential as anticoagulant drugs for the prevention and/or treatment of AML-associated DIC. Using a fluorescence-based insulin reduction assay, we confirmed inhibition of recombinant human PDI by bacitracin and quercetin-3-rutinoside (also known as rutin and recently shown to be a specific PDI inhibitor) with IC50 values of 0.6 mM and 14 μM, respectively, showing >95% inhibition at 1 mM (bacitracin) and 50 μM (rutin). Significant insulin reductase activity was observed on HL60 cells, and this activity was inhibited by 75% and 49% using 1 mM bacitracin and 100 μM rutin, respectively, suggesting the presence of additional, PDI-independent thiol isomerase activity. Short-term treatment with 100 μM rutin for 15 min also inhibited TF PCA on HL60 cells by 37%. Importantly, the inhibitory effect of rutin on cell-associated PDI and TF activity was completely abolished by cell washing, confirming previous evidence that rutin is a reversible PDI inhibitor. When HL60 cells were exposed to rutin (100 μM) for 24 hrs, cell-associated TF PCA was increased 2.3-fold (P<0.01), an effect that was accompanied by enhanced PS exposure, as assessed by annexin V-FITC binding (positive cells, 32±11 vs. 10±4%; P<0.01), and increased PCA of cellular microparticles (MPs) isolated from culture supernatants, as evidenced by the thrombin generation parameters lag phase (LP, 14±1 vs. 19±4 min), peak thrombin (PT, 55±17 vs. 22±14 nM), and area under the curve (AUC, 1193±329 vs. 476±347 nM*min; P<0.01). Interestingly, treatment with 100 μM rutin also resulted in a 1.7-fold increase in total cellular TF antigen (P=0.07). The effects of long-term incubation with bacitracin (1 mM) were even more pronounced, involving an 8.3-fold and 4.6-fold increase in cell-associated TF PCA and total cellular TF antigen, respectively. PS exposure (45±9%) and shedding of procoagulant MPs (LP, 7±1 min; PT, 175±49 nM; AUC, 2756±402 nM*min) were also significantly increased. While neither short-term nor long-term exposure to rutin affected TF PCA on THP1 cells, co-incubation with rutin dose-dependently (10–100 μM) inhibited daunorubicin-induced TF PCA in this cell model, an effect that could not be explained by decreased PS exposure. Importantly, both the reaction pattern of HL60 and that of THP1 cells were reproduced ex vivo using myeloblasts from AML patients. In summary, our findings suggest a highly complex and context-dependent role of PDI in leukemic-cell TF PCA expression. While short-term exposure to rutin can reversibly inhibit both PDI and TF activity, long-term exposure may result in significantly increased cellular TF PCA and MP shedding, pointing to a possible role of PDI in PS homeostasis, cytoskeleton rearrangement, and/or TF recycling. In addition, induction of leukemic-cell apoptosis and necrosis by cytotoxic drugs, which is associated with an early loss in membrane integrity and enhanced accessibility of cytoplasmic enzymes, may involve an additional role of (intracellular) PDI in the efficient presentation of TF PCA by AML blasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Critical Role of Cys168 in Noggin Protein's Biological Function

2005 ◽  
Vol 37 (3) ◽  
pp. 181-185
Author(s):  
Wei-Dong Liu ◽  
Xiang-Ling Feng ◽  
Cai-Ping Ren ◽  
Jian-Ling Shi ◽  
Xu-Yu Yang ◽  
...  
Keyword(s):  
Biological Activities ◽  
Critical Role ◽  
Ectopic Expression ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Morphogenetic Protein ◽  
Neural Cell Adhesion ◽  
A Site ◽  
The Relationship

Abstract Previous that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin's biological activities. It probably participates in the formation of an intermolecular disulfide bridge.

scholarly journals Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

2021 ◽  
Vol 14 ◽  
Author(s):  
François-Xavier Cantrelle ◽  
Anne Loyens ◽  
Xavier Trivelli ◽  
Oliver Reimann ◽  
Clément Despres ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Tau Protein ◽  
Posttranslational Modifications ◽  
Self Assembly ◽  
Critical Role ◽  
Small Peptides ◽  
C Terminus ◽  
The Impact

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

scholarly journals It's reticulated: the liver at the heart of atherosclerosis

2018 ◽  
Vol 238 (1) ◽  
pp. R1-R11 ◽  
Author(s):  
Prabhakara R Nagareddy ◽  
Sunil K Noothi ◽  
Michelle C Flynn ◽  
Andrew J Murphy
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Hepatic Inflammation ◽  
Reticulated Platelets ◽  
Molecular Pattern ◽  
Low Dose Aspirin ◽  
Risk Patients ◽  
Progression Of Atherosclerosis

Platelets play a critical role in both the initiation and progression of atherosclerosis, and even more so in the ensuing atherothrombotic complications. Low-dose aspirin remains the mainstay of antiplatelet therapy in high-risk patients by reducing the risk of myocardial ischemia, stroke or death due to cardiovascular disease. However, antiplatelet therapies lose their efficacy in people with diabetes mellitus, increasing the risk of future atherothrombotic events. The molecular mechanisms that promote platelet hyperactivity remain unclear but could be due to glycation-induced conformational changes of platelet membranes resulting in impaired aspirin entry or less-efficient acetylation/compensatory increase in COX-2 expression in newborn platelets. Emerging evidence from our laboratory and elsewhere suggest that enhanced platelet turnover (thrombopoiesis), particularly the production of immature reticulated platelets from the bone marrow, could contribute to atherosclerotic complications. We have identified a major role for neutrophil-derived S100A8/A9, a damage-associated molecular pattern, in driving reticulated thrombopoiesis by directly interacting with its receptors on Kupffer cells in the liver. In this review, we discuss the role of hepatic inflammation in driving reticulated platelet production and suggest potential targets to control their production, improve efficacy of current antiplatelet therapies and reduce the risk of atherothrombotic complications.

scholarly journals The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Victoria J. Madigan ◽  
Garrett E. Berry ◽  
Tyne O. Tyson ◽  
Dasean Nardone-White ◽  
Jonathan Ark ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Conformational Changes ◽  
Secretory Pathway ◽  
Critical Role ◽  
Calcium Ionophore ◽  
Calcium Atpase ◽  
Cellular Calcium ◽  
Golgi Compartment ◽  
Genome Transcription

ABSTRACT Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro. Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo. Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.

scholarly journals Critical role of backbone coordination in the mRNA recognition by RNA induced silencing complex

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lizhe Zhu ◽  
Hanlun Jiang ◽  
Siqin Cao ◽  
Ilona Christy Unarta ◽  
Xin Gao ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Critical Role ◽  
General Mechanism ◽  
Seed Region ◽  
Trap State ◽  
Biomolecular Systems ◽  
Target Mrna ◽  
Argonaute 2 ◽  
Dynamics Simulations

AbstractDespite its functional importance, the molecular mechanism underlying target mRNA recognition by Argonaute (Ago) remains largely elusive. Based on extensive all-atom molecular dynamics simulations, we constructed quasi-Markov State Model (qMSM) to reveal the dynamics during recognition at position 6-7 in the seed region of human Argonaute 2 (hAgo2). Interestingly, we found that the slowest mode of motion therein is not the gRNA-target base-pairing, but the coordination of the target phosphate groups with a set of positively charged residues of hAgo2. Moreover, the ability of Helix-7 to approach the PIWI and MID domains was found to reduce the effective volume accessible to the target mRNA and therefore facilitate both the backbone coordination and base-pair formation. Further mutant simulations revealed that alanine mutation of the D358 residue on Helix-7 enhanced a trap state to slow down the loading of target mRNA. Similar trap state was also observed when wobble pairs were introduced in g6 and g7, indicating the role of Helix-7 in suppressing non-canonical base-paring. Our study pointed to a general mechanism for mRNA recognition by eukaryotic Agos and demonstrated the promise of qMSM in investigating complex conformational changes of biomolecular systems.

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