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1.
Effect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasion.
Najahi-Missaoui, W, Quach, ND, Jenkins, A, Dabke, I, Somanath, PR, Cummings, BS
Pharmacology research & perspectives. 2019;(5):e00518
Abstract
P21-activated kinase-1 (PAK-1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK-1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK-1 were resistant to the cytotoxic effects of the PAK-1 inhibitor, inhibitor targeting PAK-1 activation-3 (IPA-3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK-1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA-3 for PAK-1 and the hypothesis that gene silencing of PAK-1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA-3 (SSL-IPA-3). PAK-1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK-1 correlated to the IC50 of IPA-3 as measured by MTT staining. PAK-1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU-145 and breast cancer MCF-7 cells. Decreased migration and invasion also correlated to decreased expression of E-cadherin and alterations in C-X-C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK-1 inhibition increased the cytotoxicity of IPA-3, and the cytotoxicity of SSL-IPA-3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK-1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA-3 and its liposomal formulation. These data also show the specificity of IPA-3 for PAK-1, are some of the first data suggesting that IPA-3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome-encapsulated IPA-3 in breast cancer cells.
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2.
Electrochemically Exfoliated High-Quality 2H-MoS2 for Multiflake Thin Film Flexible Biosensors.
Zhang, P, Yang, S, Pineda-Gómez, R, Ibarlucea, B, Ma, J, Lohe, MR, Akbar, TF, Baraban, L, Cuniberti, G, Feng, X
Small (Weinheim an der Bergstrasse, Germany). 2019;(23):e1901265
Abstract
2D molybdenum disulfide (MoS2 ) gives a new inspiration for the field of nanoelectronics, photovoltaics, and sensorics. However, the most common processing technology, e.g., liquid-phase based scalable exfoliation used for device fabrication, leads to the number of shortcomings that impede their large area production and integration. Major challenges are associated with the small size and low concentration of MoS2 flakes, as well as insufficient control over their physical properties, e.g., internal heterogeneity of the metallic and semiconducting phases. Here it is demonstrated that large semiconducting MoS2 sheets (with dimensions up to 50 µm) can be obtained by a facile cathodic exfoliation approach in nonaqueous electrolyte. The synthetic process avoids surface oxidation thus preserving the MoS2 sheets with intact crystalline structure. It is further demonstrated at the proof-of-concept level, a solution-processed large area (60 × 60 µm) flexible Ebola biosensor, based on a MoS2 thin film (6 µm thickness) fabricated via restacking of the multiple flakes on the polyimide substrate. The experimental results reveal a low detection limit (in femtomolar-picomolar range) of the fabricated sensor devices. The presented exfoliation method opens up new opportunities for fabrication of large arrays of multifunctional biomedical devices based on novel 2D materials.
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3.
Life inside and out: making and breaking protein disulfide bonds in Chlamydia.
Christensen, S, McMahon, RM, Martin, JL, Huston, WM
Critical reviews in microbiology. 2019;(1):33-50
Abstract
Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.
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4.
Studying Functional Disulphide Bonds by Computer Simulations.
Gräter, F, Li, W
Methods in molecular biology (Clifton, N.J.). 2019;:87-113
Abstract
Biochemical and structural data reveal important aspects of the properties and function of a protein disulphide bond. Molecular dynamics simulations can complement this experimental data and can yield valuable insights into the dynamical behavior of the disulphide bond within the protein environment. Due to the increasing accuracy of the underlying energetic description and the increasing computational power at hand, such simulations have now reached a level, at which they can also make quantitative and experimentally testable predictions. We here give an overview of the computational methods used to predict functional aspects of protein disulphides, including the prestress, protein allosteric effects upon thiol/disulphide exchange, and disulphide redox potentials. We then outline in detail the use of free-energy perturbation methods to calculate the redox potential of a protein disulphide bond of interest. In a step-by-step protocol, we describe the workflow within the MD suite Gromacs, including practical advice on the simulation setup and choice of parameters. For other disulphide-related simulation methods, we refer to resources available online.
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5.
Effects of the anesthesia technique used during cesarean section on maternal-neonatal thiol disulfide homeostasis.
Akin, F, Kozanhan, B, Deniz, CD, Sahin, O, Goktepe, H, Neselioglu, S, Erel, O
Minerva anestesiologica. 2019;(11):1175-1183
Abstract
BACKGROUND Thiols are organic compounds consisting of a sulfhydryl group which exerts antioxidant effects via dynamic thiol-disulfide homeostasis (TDH). The shift towards the disulfide states signals an oxidative situation. Maternal-neonatal oxidative stress can be affected by the anesthetic technique used during cesarean section. This study aimed to evaluate the relationship between the type of anesthesia used and the maternal-neonatal TDH. METHODS ASA I-II, term parturients undergoing elective cesarean section either under general (Group G) or spinal (Group S) anesthesia were included. Blood specimens were collected preoperatively and postoperatively from the mothers and from the umbilical venous cords at delivery. TDH was studied by a new method developed by the authors (O.E. and S.N.). RESULTS Postoperative mother's native thiol, total thiol, disulfide/total thiol, native thiol/ disulfide and disulfide levels were higher in Group G than in Group S. There was no significant difference between the groups in umbilical venous cord albumin, native thiol/total thiol, disulfide, native thiol/ disulfide and total thiol/ disulfide. However, in Group G, umbilical venous cord native thiol and total thiol levels were statistically significantly lower than those in Group S. CONCLUSIONS Our results showed that general anesthesia in cesarean section leads to an impairment in TDH when compared with spinal anesthesia. Oxidative stress might be modified by the preferred anesthetic technique.
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6.
Applications of catalyzed cytoplasmic disulfide bond formation.
Saaranen, MJ, Ruddock, LW
Biochemical Society transactions. 2019;(5):1223-1231
Abstract
Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.
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7.
Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder.
Singh, S, Akhter, MS, Dodt, J, Sharma, A, Kaniyappan, S, Yadegari, H, Ivaskevicius, V, Oldenburg, J, Biswas, A
International journal of molecular sciences. 2019;(8)
Abstract
Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.
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8.
A Near-Infrared-Controllable Artificial Metalloprotease Used for Degrading Amyloid-β Monomers and Aggregates.
Ma, M, Wang, Y, Gao, N, Liu, X, Sun, Y, Ren, J, Qu, X
Chemistry (Weinheim an der Bergstrasse, Germany). 2019;(51):11852-11858
Abstract
Proteolysis of amyloid-β (Aβ) is a promising approach against Alzheimer's disease. However, it is not feasible to employ natural hydrolases directly because of their cumbersome preparation and purification, poor stability, and hazardous immunogenicity. Therefore, artificial enzymes have been developed as potential alternatives to natural hydrolases. Since specific cleavage sites of Aβ are usually embedded inside the β-sheet structures that restrict access by artificial enzymes, this strongly hinders their efficiency for practical applications. Herein, we construct a NIR (near-IR) controllable artificial metalloprotease (MoS2 -Co) using a molybdenum disulfide nanosheet (MoS2 ) and a cobalt complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Codota). Evidenced by detailed experimental and theoretical studies, the NIR-enhanced MoS2 -Co can circumvent the restriction by simultaneously inhibition of β-sheet formation and destroying β-sheet structures of the preformed Aβ aggregates in living cell. Furthermore, our designed MoS2 -Co is an easy to graft Aβ-target agent that prevents misdirected or undesirable hydrolysis reactions, and has been demonstrated to cross the blood brain barrier. This method can be adapted for hydrolysis of other kinds of amyloids.
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9.
Engineering of the Dsb (disulfide bond) proteins - contribution towards understanding their mechanism of action and their applications in biotechnology and medicine.
Banaś, AM, Bocian-Ostrzycka, KM, Jagusztyn-Krynicka, EK
Critical reviews in microbiology. 2019;(4):433-450
Abstract
The Dsb protein family in prokaryotes catalyzes the generation of disulfide bonds between thiol groups of cysteine residues in nascent proteins, ensuring their proper three-dimensional structure; these bonds are crucial for protein stability and function. The first Dsb protein, Escherichia coli DsbA, was described in 1991. Since then, many details of the bond-formation process have been described through microbiological, biochemical, biophysical and bioinformatics strategies. Research with the model microorganism E. coli and many other bacterial species revealed an enormous diversity of bond-formation mechanisms. Research using Dsb protein engineering has significantly helped to reveal details of the disulfide bond formation. The first part of this review presents the research that led to understanding the mechanism of action of DsbA proteins, which directly transfer their own disulfide into target proteins. The second part concentrates on the mechanism of electron transport through the cell cytoplasmic membrane. Third and lastly, the review discusses the contribution of this research towards new antibacterial agents.
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10.
Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.
Le, ST, Guros, NB, Bruce, RC, Cardone, A, Amin, ND, Zhang, S, Klauda, JB, Pant, HC, Richter, CA, Balijepalli, A
Nanoscale. 2019;(33):15622-15632
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Abstract
We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.