Subhabrata Majumder

In Vivo Activation of the p53 Tumor Suppressor Pathway by an Engineered Cyclotide.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50≈20 nM) and an efficient HIV-1 cell-entry blocker (EC50≈2 nM). This cyclotide also showed high stability in human serum, thereby providing a promising lead compound for the design of a novel type of peptide-based anticancer and anti-HIV-1 therapeutics.

Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide–Protein Interactions†

Cyclotides are fascinating natural plant micro-proteins ranging from 28 to 37 amino acid residues long and exhibit various biological actions including anti-microbial, insecticidal, cytotoxic, antiviral (against HIV), protease inhibitory, and hormone-like activities.[1–4] They share a unique head-to-tail circular knotted topology of three disulfide bridges; one disulfide penetrates through a macrocycle formed by the other two disulfides, thereby inter-connecting the peptide backbone to form what is called a cystine knot topology (Fig. 1). This cyclic cystine knot (CCK) framework gives the cyclotides exceptional rigidity[5], resistance to thermal and chemical denaturation, and enzymatic stability against degradation.[4, 6] In fact, some cyclotides have been shown to be orally bioavailable. For example, the first cyclotide to be discovered, kalata B1, was found to be an orally effective uterotonic,[7] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[8–10] All of these features make cyclotides ideal tools for drug development.[11–14]

Probing Protein Quinary Interactions by In-Cell Nuclear Magnetic Resonance Spectroscopy

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs.

Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker′s Yeast†

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

Recombinant production of rhesus θ-defensin-1 (RTD-1) using a bacterial expression system

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus θ-defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds and an unusual backbone cyclic topology. In this work we report for the first time the recombinant expression of the fully folded θ-defensin RTD-1 using a bacterial expression system. This was accomplished using an intramolecular native chemical ligation in combination with a modified protein-splicing unit. RTD-1 was produced either in vitro or in vivo. In-cell production of RTD-1 was estimated to reach an intracellular concentration of ~4 μM. Recombinant RTD-1 was shown to be correctly folded as characterized by heteronuclear-NMR and by its ability to specifically inhibit lethal factor protease. The recombinant production of folded θ-defensins opens the possibility to produce peptide libraries based on this peptide scaffold that could be used to develop in-cell screening and directed evolution technologies.

Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1).

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.

Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity

We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin (1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based in the treatment of cancer and myocardial infarction.

Using singular value decomposition to characterize protein-protein interactions by in-cell NMR spectroscopy.

Distinct differences between how model proteins interact in‐cell and in vitro suggest that the cytosol might have a profound effect in modulating protein–protein and/or protein–ligand interactions that are not observed in vitro. Analyses of in‐cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal‐to‐noise ratios, and the long overexpression times used in protein–protein interaction studies may lead to changes in the in‐cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in‐cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single‐value decomposition (SVD). SVD differentiates between concentration‐dependent and concentration‐independent events and identifies the principal binding mode between the two species. The analysis implicates a set of amino acids involved in the specific interaction that differs from previous NMR analyses but is in good agreement with crystallographic data.

Total Cellular RNA Modulates Protein Activity

RNA constitutes up to 20% of a cells dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.