Ishu Saraogi

Recent advances in the development of aryl-based foldamers

In recent years, significant effort has gone into making synthetic oligomers that can attain well-defined conformations analogous to the folding of biomolecules. The diversity of the structural building blocks ranges from peptidic and other aliphatic repeat units to aromatic ones, which do not have a natural counterpart. In this critical review, we will focus on the developments in aromatic foldamers in the last two years and their potential applications. This review will be of interest to people working on the structural and functional mimicry of biomolecules and will, we hope, stimulate further research into novel applications (149 references).

Synthetic α-Helix Mimetics as Agonists and Antagonists of Islet Amyloid Polypeptide Aggregation

The development of small molecules that can modulate the damaging effects of protein aggregation processes remains a high priority goal in contemporary medicinal chemistry.[1] An important class of these aggregates, called amyloids, has been implicated in numerous degenerative diseases including Alzheimer’s, type II diabetes, senile systemic amyloidosis (SSA), prion diseases and rheumatoid arthritis. The attribute shared by these symptomatically unrelated diseases is that a normally soluble protein undergoes a conformational change resulting in self-assembly into cytotoxic forms culminating in a β-sheet rich fibrillar structure. Islet amyloid polypeptide (IAPP), or amylin, is one such protein that has been implicated in amyloidogenesis in type II diabetes.[2] IAPP is cosecreted with insulin by the β-cells of the islets of Langerhans and an aggregated form of IAPP is believed to play a role in β-cell toxicity in the pathology of type II diabetes.[3]

A Peptidomimetic Approach to Targeting Pre-amyloidogenic States in Type II Diabetes

Protein fiber formation is associated with diseases ranging from Alzheimers to type II diabetes. For many systems, including islet amyloid polypeptide (IAPP) from type II diabetes, fibrillogenesis can be catalyzed by lipid bilayers. Paradoxically, amyloid fibers are beta sheet rich while membrane-stabilized states are alpha-helical. Here, a small molecule alpha helix mimetic, IS5, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP-induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative alpha-helical region of IAPP, revealing that alpha-helical states are on pathway to fiber formation. IAPP is not normally amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.

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.

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. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

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. A series of small molecule, α-helical mimetics, oligopyridylamides, was previously shown to target the membrane-bound α-helical oligomeric intermediates of IAPP. In this study, we develop an improved, microwave-assisted synthesis of oligopyridylamides. A series of designed tripyridylamides demonstrate that lipid-catalyzed self-assembly of IAPP can be deliberately targeted. In addition, these molecules affect IAPP-induced leakage of synthetic liposomes and cellular toxicity in insulin-secreting cells. The tripyridylamides inhibit these processes with identical rank orders of effectiveness. This indicates a common molecular basis for the disparate set of observed effects of IAPP.

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. Collectively, these studies elucidate how an essential SRP RNA and two regulatory GTPases in the SRP and SRP receptor (SR) enable this targeting machinery to recognize, sense and respond to its biological effectors, i.e. the cargo protein, the target membrane and the translocation machinery, thus driving efficient and faithful co-translational protein targeting. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

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

Active and sequential regulation of the interaction of SRP with translating ribosomes drives efficient and faithful cotranslational protein targeting to the target membrane.

Surface Binding Inhibitors of the SCF–KIT Protein–Protein Interaction

KIT is a receptor tyrosine kinase (RTK), the interaction of which with its ligand, stem cell factor (SCF), is essential for growth and differentiation of various cells.[1] SCF binding promotes KIT dimerization,[2] transphosphorylation, and activation of downstream cell signaling pathways essential for cell proliferation, differentiation, and survival. Gain-of-function mutations in KIT have been identified in human cancers such as gastrointestinal stromal tumors (GIST).[3,4] It was also demonstrated that autocrine or paracrine mechanisms mediated by aberrant expression of SCF and/or KIT might also lead to oncogenesis.[5–7] Because most cases of GIST are driven by oncogenic KIT mutations resulting in enhanced tyrosine kinase activity, inhibitors of the tyrosine kinase[8] activity of KIT, such as Gleevec® (imatinib mesylate) and Sutent® (sunitinib), have been successfully applied for the treatment of GIST patients.

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Significance Correct protein biogenesis is crucial for all cells. Numerous factors including molecular chaperones, modification enzymes, and protein-targeting machineries bind near the ribosome exit site and can access the nascent protein. How nascent proteins are accurately selected into the correct biogenesis pathway in such a crowded environment is an emerging question central to accurate protein biogenesis. Using chemical biology and biochemical and biophysical tools, we show that the major cotranslational chaperone, trigger factor, and cotranslational targeting machinery, signal recognition particle, regulate each other at multiple stages, including initial binding, ribosome delivery to the membrane, and enforcement of a timer for cotranslational protein targeting. Together, these mechanisms enhance accurate substrate selection into both cotranslational and posttranslational protein targeting pathways. 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). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.