Harnessing RNA-Protein Interactions for Therapeutic Interventions.

Interactions between RNAs and proteins play a crucial role in various diseases, including viral infections and cancer. Hence, understanding and inhibiting these interactions are important for the development of novel therapeutics. However, the identification of drugs targeting RNA-protein interactions with high specificity and affinity is challenged by our limited molecular understanding of these interactions.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.

Bacterial GTPases as druggable targets to tackle antimicrobial resistance.

Small molecules as antibacterial agents have contributed immensely to the growth of modern medicine over the last several decades. However, the emergence of drug resistance among bacterial pathogens has undermined the effectiveness of the existing antibiotics. Thus, there is an exigency to address the antibiotic crisis by developing new antibacterial agents and identifying novel drug targets in bacteria.

Dual Role of a Fluorescent Small Molecule as a Sensor and Inhibitor of Protein Fibrillation.

Ordered fibrillar aggregates of proteins, called amyloids, are prevalent in several diseases like Alzheimer's, Parkinson's, and Type II diabetes. The key challenge in the treatment of such diseases is the early detection of protein fibrillation and its effective inhibition using extrinsic agents. Thus, molecules that can both detect and inhibit protein fibril formation have great diagnostic and therapeutic utility.

Investigating the functional role of a buried interchain aromatic cluster in Escherichia coli GrpE dimer.

Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer.

An amphiphilic small molecule drives insulin aggregation inhibition and amyloid disintegration.

The aggregation of proteins into ordered fibrillar structures called amyloids, and their disintegration represent major unsolved problems that limit the therapeutic applications of several proteins. For example, insulin, commonly used for the treatment of diabetes, is susceptible to amyloid formation upon exposure to non-physiological conditions, resulting in a loss of its biological activity. Here, we report a novel amphiphilic molecule called PAD-S, which acts as a chemical chaperone and completely inhibits fibrillation of insulin and its biosimilars.

Enhanced Codon-Anticodon Interaction at In-Frame UAG Stop Codon Improves the Efficiency of Non-Natural Amino Acid Mutagenesis.

The introduction of non-natural amino acids into proteins through the stop codon readthrough methodology has been used to design proteins for diverse applications. However, this method suffers from low yields of the modified protein, as the suppressor tRNA that recognizes the stop codon is unable to compete effectively with release factor 1 (RF1), which terminates translation. We reasoned that a suppressor tRNA with improved interaction with the UAG stop codon on the mRNA will be able to compete more effectively with RF1.

Molecular Aspects of Insulin Aggregation and Various Therapeutic Interventions.

Protein aggregation leading to the formation of amyloid fibrils has various adverse effects on human health ranging from fatigue and numbness to organ failure and death in extreme cases. Insulin, a peptide hormone commonly used to treat diabetes, undergoes aggregation at the site of repeated injections in diabetic patients as well as during its industrial production and transport. The reduced bioavailability of insulin due to aggregation hinders the proper control of glucose levels in diabetic patients.

Novel Antibacterial Targets in Protein Biogenesis Pathways.

Antibiotic resistance has emerged as a global threat due to the ability of bacteria to quickly evolve in response to the selection pressure induced by anti-infective drugs. Thus, there is an urgent need to develop new antibiotics against resistant bacteria. In this review, we discuss pathways involving bacterial protein biogenesis as attractive antibacterial targets since many of them are essential for bacterial survival and virulence.

Synthesis and Evaluation of Arylamides with Hydrophobic Side Chains for Insulin Aggregation Inhibition.

Insulin, a peptide hormone, forms fibrils under aberrant physiological conditions leading to a reduction in its biological activity. To ameliorate insulin aggregation, we have synthesized a small library of oligopyridylamide foldamers decorated with different combination of hydrophobic side chains. Screening of these compounds for insulin aggregation inhibition using a Thioflavin-T assay resulted in the identification of a few hit molecules.

A Stutter in the Coiled-Coil Domain of Co-chaperone GrpE Connects Structure with Function.

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK.

Amphiphilic Small-Molecule Assemblies to Enhance the Solubility and Stability of Hydrophobic Drugs.

Amphiphilic assemblies made from diverse synthetic building blocks are well known for their biomedical applications. Here, we report the synthesis of gemini-type amphiphilic molecules that form stable assemblies in water. The assembly property of molecule M2 in aqueous solutions was first inferred from peak broadening observed in the proton NMR spectrum.

Selective functionalization at N-position of guanine in oligonucleotides via reductive amination.

Chemo- and site-specific modifications in oligonucleotides have wide applicability as mechanistic probes in chemical biology. However, methods that label specific sites in nucleic acids are scarce, especially for labeling DNA/RNA from biological or enzymatic sources rather than synthetic ones. Here we have employed a classical reaction, reductive amination, to selectively functionalize the N-amine of guanosine and 2'-deoxyguanosine monophosphate (GMP/dGMP).

Biocompatible Fluorescent Probe for Selective Detection of Amyloid Fibrils.

The misfolding and aggregation of proteins leading to amyloid formation has been linked to numerous diseases, necessitating the development of tools to monitor the fibrillation process. Here, we report an intramolecular charge transfer (ICT) dye, DMNDC, as an alternative to thioflavin-T (ThT), most commonly used for monitoring amyloid fibrils. Using insulin as a model protein, we show that DMNDC efficiently reports on the kinetics of fibril formation.

Small-Molecule Inhibitor Prevents Insulin Fibrillogenesis and Preserves Activity.

Amyloidosis is a well-known but poorly understood phenomenon caused by the aggregation of proteins, often leading to pathological conditions. For example, the aggregation of insulin poses significant challenges during the preparation of pharmaceutical insulin formulations commonly used to treat diabetic patients. Therefore, it is essential to develop inhibitors of insulin aggregation for potential biomedical applications and for important mechanistic insights into amyloidogenic pathways.

Peptide nucleic acid mediated inhibition of the bacterial signal recognition particle.

We have identified the bacterial signal recognition particle (SRP) as a novel antibacterial target. As a proof of principle, we used an antisense peptide nucleic acid to target a key SRP RNA. The PNA molecules showed efficient inhibition of SRP function and bacterial cell growth, thereby validating our hypothesis.

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting.

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP).

Islet amyloid-induced cell death and bilayer integrity loss share a molecular origin targetable with oligopyridylamide-based α-helical mimetics.

Islet amyloid polypeptide (IAPP) is a hormone cosecreted with insulin. IAPP proceeds through a series of conformational changes from random coil to β-sheet via transient α-helical intermediates. An unknown subset of these events are associated with seemingly disparate gains of function, including catalysis of self-assembly, membrane penetration, loss of membrane integrity, mitochondrial localization, and finally, cytotoxicity, a central component of diabetic pathology.

Regulation of cargo recognition, commitment, and unloading drives cotranslational protein targeting.

Efficient and accurate protein localization is essential to cells and requires protein-targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway.

Co-translational protein targeting to the bacterial membrane.

Co-translational protein targeting by the Signal Recognition Particle (SRP) is an essential cellular pathway that couples the synthesis of nascent proteins to their proper cellular localization. The bacterial SRP, which contains the minimal ribonucleoprotein core of this universally conserved targeting machine, has served as a paradigm for understanding the molecular basis of protein localization in all cells. In this review, we highlight recent biochemical and structural insights into the molecular mechanisms by which fundamental challenges faced by protein targeting machineries are met in the SRP pathway.

Fingerloop activates cargo delivery and unloading during cotranslational protein targeting.

During cotranslational protein targeting by the signal recognition particle (SRP), information about signal sequence binding in the SRP's M domain must be effectively communicated to its GTPase domain to turn on its interaction with the SRP receptor (SR) and thus deliver the cargo proteins to the membrane. A universally conserved "fingerloop" lines the signal sequence-binding groove of SRP; the precise role of this fingerloop in protein targeting has remained elusive. In this study, we show that the fingerloop plays important roles in SRP function by helping to induce the SRP into a more active conformation that facilitates multiple steps in the pathway, including efficient recruitment of SR, GTPase activation in the SRP•SR complex, and most significantly, the unloading of cargo onto the target membrane.

A tale of two GTPases in cotranslational protein targeting.

Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The "GTPase switch" paradigm, in which a GTPase acts as a bimodal switch that is turned "on" and "off" by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation.

Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide.

Molecular mechanism of co-translational protein targeting by the signal recognition particle.

The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. The SRP pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membrane, the productive unloading of cargo to the translocation machinery and the precise spatial and temporal coordination of these molecular events. Here we highlight recent advances in our understanding of the molecular mechanisms underlying this pathway, and discuss new questions raised by these findings.