Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase

<p>Abstract</p> <p>Background</p> <p>In contrast to many other organisms, the malarial parasite <it>Plasmodium falciparum </it>possesses only one typical glutathione <it>S</it>-transferase. This enzyme, <it>Pf</it>GST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The <it>Pf</it>GST under native conditions forms non-covalently linked higher aggregates with major population (~98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of <it>Pf</it>GST is observed. Recently reported study on binding and catalytic properties of <it>Pf</it>GST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to <it>Pf</it>GST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of <it>Pf</it>GST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant <it>Pf</it>GST in the presence and absence of GSH were carried out.</p> <p>Results</p> <p>Our data indicate that the dimer – and not the tetramer – is the active form of <it>Pf</it>GST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of <it>Pf</it>GST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the <it>Pf</it>GST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it.</p> <p>Conclusion</p> <p>This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of <it>Plasmodium falciparum </it>glutathione <it>S</it>-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers.</p

Autophagy modulation as a treatment of amyloid diseases

Amyloids are fibrous proteins aggregated into toxic forms that are implicated in several chronic disorders. More than 30 diseases show deposition of fibrous amyloid proteins associated with cell loss and degeneration in the affected tissues. Evidence demonstrates that amyloid diseases result from protein aggregation or impaired amyloid clearance, but the connection between amyloid accumulation and tissue degeneration is not clear. Common examples of amyloid diseases are Alzheimer\u27s disease (AD), Parkinson\u27s disease (PD) and tauopathies, which are the most common forms of neurodegenerative diseases, as well as polyglutamine disorders and certain peripheral metabolic diseases. In these diseases, increased accumulation of toxic amyloid proteins is suspected to be one of the main causative factors in the disease pathogenesis. It is therefore important to more clearly understand how these toxic amyloid proteins accumulate as this will aide in the development of more effective preventive and therapeutic strategies. Protein homeostasis, or proteostasis, is maintained by multiple cellular pathways-including protein synthesis, quality control, and clearance-which are collectively responsible for preventing protein misfolding or aggregation. Modulating protein degradation is a very complex but attractive treatment strategy used to remove amyloid and improve cell survival. This review will focus on autophagy, an important clearance pathway of amyloid proteins, and strategies for using it as a potential therapeutic target for amyloid diseases. The physiological role of autophagy in cells, pathways for its modulation, its connection with apoptosis, cell models and caveats in developing autophagy as a treatment and as a biomarker is discussed

An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

Bacterial cells can be engineered to express non-native genes, resulting in the production of, recombinant proteins, which have various biotechnological and pharmaceutical applications. In eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression system, although the expression is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG), studies have shown low expression levels of proteins. Irrespective of the purpose of protein production, the production process requires the accomplishment of three individual factors: expression, solubilization and purification. Although several efforts, including changing the host, vector, culture parameters of the recombinant host strain, co-expression of other genes and changing of the gene sequences, have been directed towards enhancing recombinant protein expression, the protein expression is still considered as a significant limiting step. Our protocol explains a simple method to enhance the recombinant protein expression that we have optimized using several unrelated proteins. It works with both T5 and T7 promoters. This protocol can be used to enhance the expressions of most of the proteins. The advantages of this technique are presented below:• It produces several fold increase in the expression of poorly expressed, less expressed or non-expressed recombinant proteins. • It does not employ any additional component such as chaperones, heat shock proteins or co-expression of other genes. • In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system

How Does Arbidol Inhibit the Novel Coronavirus SARS-CoV-2? Atomistic Insights from Molecular Dynamics Simulations

The COVID-19 pandemic is spreading at an alarming rate, posing an unprecedented threat to the global economy and human health. Broad-spectrum antivirals are currently being administered for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) treatment. China\u27s prevention and treatment guidelines suggest the use of an anti-influenza drug, Arbidol, for the clinical treatment of COVID-19. Reports indicate that Arbidol could neutralize the SARS-CoV-2. Monotherapy with Arbidol is found superior to Lopinavir-Ritonavir or Favipiravir in the treatment of COVID-19. In the SARS-CoV-2, Arbidol acts upon interfering in virus binding to host cells. However, the detailed understanding of Arbidol induced inhibition of SARS-CoV-2 is not known. Here, we present atomistic insights into the Arbidol-induced SARS-CoV-2 membrane fusion inhibition and propose a model of inhibition. Molecular dynamics (MD) simulation-based analyses demonstrate that Arbidol binds and stabilizes at the receptor-binding domain (RBD)/ACE2 interface with a high affinity. It forms stronger intermolecular interactions with RBD than ACE2. Analyses of the detailed decomposition of energy components and binding affinities revealed a substantial increase in the affinity between RBD and ACE2 in the Arbidol-bound RBD/ACE2 complex, suggesting that Arbidol could generate favorable interactions between them. Based on our MD simulation results, we propose that the binding of Arbidol induced structural rigidity in the virus glycoprotein resulting in restriction of the conformational rearrangements associated with membrane attachment and virus entry.Further, key residues of RBD and ACE2 that interacted with Arbidol were identified, opening the doors for the development of therapeutic strategies and higher efficacy Arbidol derivatives or lead drug candidates.</p

Structural and stability characteristics of a monothiol glutaredoxin: glutaredoxin-like protein 1 from Plasmodium falciparum

Recently discovered monothiol glutaredoxins with CXXS-active site sequence share a common structural motif and biochemical mechanism of action and are involved in multiple cellular functions. Here we report first studies on the structural and stability characterization of a monothiol glutaredoxin, in particular - PfGLP1. Our results demonstrate that in the native conformation, the enzyme has a compact core structure with a relatively flexible N-terminal portion having an open configuration. Comparative functional studies with the full-length and N-terminal truncated protein demonstrate that the flexible N-terminal portion does not play any significant role in functional activity of the protein. In contrast to other Grxs, PfGLP1 does not contain a Fe-S cluster. The pH dependent studies demonstrate that the protein is resistant to alkaline pH but highly sensitive to acidic pH and undergoes significant unfolding between pH 4 and 5. However, acidic conditions also do not induce complete unfolding of the enzyme. The protein is stabilized with a conformational free energy of about 3.2 &#177; 0.1 kcal mol- 1. The protein is a highly cooperative molecule as during denaturant-induced equilibrium unfolding a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed

Conformational stability and energetics of Plasmodium falciparum glutaredoxin

Glutaredoxins (Grxs), redox-active proteins with a typical -CPYC motif at their active sites, are involved in redox-regulatory processes and antioxidant defenses. The human malarial parasite Plasmodium falciparum possess a classical glutaredoxin (PfGrx) as well as a number of Grx-like proteins. In the present study, we investigated the unfolding energetics and conformational stability of PfGrx, using isothermal guanidine hydrochloride-induced and pH-dependent thermal denaturation. Reversible unfolding can be modeled using a two-state transition between the native and unfolded states. The structural topology of the protein was stable over a wide pH range from 3.0 to 11.0. Although the protein was thermally stable, it exhibited a small free energy of 1.56 kcal mol-1 at 25&#176;C. The thermostability of PfGrx reached its maximum at pH 8.0, with a Tm of 76.2 &#176;C and a &#916;Hm of 119 kcal mol-1. To elucidate the factors underlying the thermostability, a protein stability curve was generated. Maximum stability occurred at around 47 &#176;C, where the &#916;GDH2O value was 4.30 kcal mol-1. The high structural stability over a broad pH range, together with the capacity to endure very high temperatures, supports the notion that Grx can withstand a wide variety of conditions, allowing it to play a key role in cellular redox homeostasis. To the best of our knowledge, this work represents the first attempt to understand the energetic characteristics of a glutaredoxin in relation to accompanying structural changes