scholarly journals Mapping the substrate sequence and length of the Plasmodium M1 and M17 aminopeptidases

2020 ◽  
Author(s):  
Tess R Malcolm ◽  
Karolina W. Swiderska ◽  
Brooke K Hayes ◽  
Marcin Drag ◽  
Nyssa Drinkwater ◽  
...  
Keyword(s):  
Drug Targets ◽  
Protein Production ◽  
Behavioral Responses ◽  
Peptide Sequence ◽  
Sequence Specificity ◽  
Model Species ◽  
Hydrophobic Residues ◽  
Digestion Process ◽  
Malarial Infection ◽  
Peptide Length

AbstractDuring malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dualtarget anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. When tested with a panel of peptides of increasing length, each aminopeptidase exhibited unique catalytic behavioral responses to the increase in peptide length, although all six aminopeptidases exhibited an increase in cooperativity as peptide length increased. Overall the P. vivax and P. berghei enzymes were generally faster than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

scholarly journals Mapping the substrate specificity of the Plasmodium M1 and M17 aminopeptidases.

2021 ◽  
Author(s):  
Tess R Malcolm ◽  
Karolina W. Swiderska ◽  
Brooke K Hayes ◽  
Chaille T Webb ◽  
Marcin Drag ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Drug Targets ◽  
Protein Production ◽  
Turnover Rate ◽  
Plasmodium Species ◽  
Peptide Sequence ◽  
Sequence Specificity ◽  
Model Species ◽  
Hydrophobic Residues ◽  
Digestion Process

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

Small Molecules Effective Against Liver and Blood Stage Malarial Infection

2019 ◽  
Vol 18 (23) ◽  
pp. 2008-2021 ◽  
Author(s):  
Snigdha Singh ◽  
Neha Sharma ◽  
Charu Upadhyay ◽  
Sumit Kumar ◽  
Brijesh Rathi ◽  
...  
Keyword(s):  
Drug Targets ◽  
Malaria Parasite ◽  
Blood Stage ◽  
Culture Methods ◽  
Liver Stage ◽  
Malarial Infection ◽  
Dual Stage ◽  
New Treatment ◽  
Global Threat

Malaria is a lethal disease causing devastating global impact by killing more than 8,00,000 individuals yearly. A noticeable decline in malaria related deaths can be attributed to the most reliable treatment, ACTs against P. falciparum. However, the cumulative resistance of the malaria parasite against ACTs is a global threat to control the disease and, therefore the new effective therapeutics are urgently needed, including new treatment approaches. Majority of the antimalarial drugs target BS malarial infection. Currently, scientists are eager to explore the drugs with potency against not only BS but other life stages such as sexual and asexual stages of the malaria parasite. Liver Stage is considered as one of the important drug targets as it always leads to BS and the infection can be cured at this stage before it enters into the Blood Stage. However, a limited number of compounds are reported effective against LS malaria infection probably due to scarcity of in vitro LS culture methods and clinical possibilities. This mini review covers a range of chemical compounds showing efficacy against BS and LS of the malaria parasite’s life cycle collectively (i.e. dual stage activity). These scaffolds targeting dual stages are essential for the eradication of malaria and to evade resistance.

Bimodal evolution of Src and Abl kinase substrate specificity revealed using mammalian cell extract as substrate pool

2020 ◽  
Author(s):  
Patrick Finneran ◽  
Margaret Soucheray ◽  
Christopher Wilson ◽  
Renee Otten ◽  
Vanessa Buosi ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Mammalian Cell ◽  
Tyrosine Kinases ◽  
Cell Extract ◽  
Peptide Sequence ◽  
Last Common Ancestor ◽  
Sequence Specificity ◽  
Kinase Substrate ◽  
Evolutionary Trajectories ◽  
Substrate Proteins

AbstractThe specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. While peptide sequence specificity for individual kinases has been examined previously, here we explore the evolutionary progression that has led to the modern substrate specificity of two non-receptor tyrosine kinases, Abl and Src. To efficiently determine the substrate specificity of modern and reconstructed ancestral kinases, we developed a method using mammalian cell lysate as the substrate pool, thereby representing the naturally occurring substrate proteins. We find that the oldest tyrosine kinase ancestor was a promiscuous enzyme that evolved through a more specific last common ancestor into a specific human Abl. In contrast, the parallel pathway to human Src involved a loss of substrate specificity, leading to general promiscuity. These results add a new facet to our understanding of the evolution of signaling pathways, with both subfunctionalization and neofunctionalization along the evolutionary trajectories.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

scholarly journals Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

2015 ◽  
Vol 197 (19) ◽  
pp. 3133-3141 ◽  
Author(s):  
Olga Bantysh ◽  
Marina Serebryakova ◽  
Inna Zukher ◽  
Alexey Kulikovsky ◽  
Darya Tsibulskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Functional Characterization ◽  
Trna Synthetase ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Target Cells ◽  
Content Type ◽  
Peptide Length

ABSTRACTEscherichia colimicrocin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitiveEscherichia colistrains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions ofE. coliMccA heptapeptide do not affect MccB-catalyzed adenylation and that some extended-peptide-length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose-binding protein (MBP) with the MccA peptide is also recognized by MccBin vivoandin vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins.IMPORTANCEEnzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature.

scholarly journals Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

2009 ◽  
Vol 75 (23) ◽  
pp. 7356-7364 ◽  
Author(s):  
Jessica C. Zweers ◽  
Thomas Wiegert ◽  
Jan Maarten van Dijl
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Protein Production ◽  
Cell Envelope ◽  
Regulatory System ◽  
Interesting Candidate ◽  
High Level ◽  
And Function

ABSTRACT Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the σW regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

scholarly journals Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria

2003 ◽  
Vol 47 (9) ◽  
pp. 2875-2881 ◽  
Author(s):  
R. Edward Benson ◽  
Elizabeth B. Gottlin ◽  
Dale J. Christensen ◽  
Paul T. Hamilton
Keyword(s):  
Cell Growth ◽  
Phage Display ◽  
Drug Targets ◽  
Antimicrobial Agents ◽  
Specific Interaction ◽  
Screening Tools ◽  
Peptide Sequence ◽  
Essential Proteins ◽  
Intracellular Expression ◽  
Protein Essentiality

ABSTRACT We describe a “protein knockout” technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

scholarly journals In Silico Proteome Cleavage Reveals Iterative Digestion Strategy for High Sequence Coverage

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jesse G. Meyer
Keyword(s):  
Posttranslational Modifications ◽  
Rna Splicing ◽  
Shotgun Proteomics ◽  
Peptide Sequence ◽  
Sequence Coverage ◽  
Proteome Coverage ◽  
Protein Inference ◽  
Peptide Length ◽  
Splicing Variants

In the postgenome era, biologists have sought to measure the complete complement of proteins, termed proteomics. Currently, the most effective method to measure the proteome is with shotgun, or bottom-up, proteomics, in which the proteome is digested into peptides that are identified followed by protein inference. Despite continuous improvements to all steps of the shotgun proteomics workflow, observed proteome coverage is often low; some proteins are identified by a single peptide sequence. Complete proteome sequence coverage would allow comprehensive characterization of RNA splicing variants and all posttranslational modifications, which would drastically improve the accuracy of biological models. There are many reasons for the sequence coverage deficit, but ultimately peptide length determines sequence observability. Peptides that are too short are lost because they match many protein sequences and their true origin is ambiguous. The maximum observable peptide length is determined by several analytical challenges. This paper explores computationally how peptide lengths produced from several common proteome digestion methods limit observable proteome coverage. Iterative proteome cleavage strategies are also explored. These simulations reveal that maximized proteome coverage can be achieved by use of an iterative digestion protocol involving multiple proteases and chemical cleavages that theoretically allow 92.9% proteome coverage.

Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain

2017 ◽  
Vol 474 (16) ◽  
pp. 2817-2828 ◽  
Author(s):  
Yitian Xia ◽  
Yuan Shang ◽  
Rongguang Zhang ◽  
Jinwei Zhu
Keyword(s):  
Conformational Transition ◽  
Target Recognition ◽  
Complex Structure ◽  
Scaffold Proteins ◽  
Peptide Sequence ◽  
Structural Comparison ◽  
Dynamic Regulation ◽  
Hydrophobic Residues ◽  
Recognition Specificity ◽  
Hydrophobic Site

The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the ‘hydrophobic site’ of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the ‘phosphor-site’ of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs.

scholarly journals Malaria parasite plasmepsins: More than just plain old degradative pepsins

2020 ◽  
Vol 295 (25) ◽  
pp. 8425-8441 ◽  
Author(s):  
Armiyaw S. Nasamu ◽  
Alexander J. Polino ◽  
Eva S. Istvan ◽  
Daniel E. Goldberg
Keyword(s):  
Drug Targets ◽  
Malaria Parasite ◽  
Sequence Specificity ◽  
Digestive Vacuole ◽  
Aspartic Proteases ◽  
Transmission Stage ◽  
Parasite Biology ◽  
Hemoglobin Degradation ◽  
Key Aspects ◽  
Antimalarial Chemotherapy

Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium. Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism.

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