Biology subjects

Rajasekaran, N.

Publications and source records attributed to Rajasekaran, N..

5 recordsLinked to original sources

bioRxiv2026

Multivalent weak contacts shape chaperone-nascent protein interactions

Molecular chaperones interact with non-native proteins, playing crucial roles in preventing misfolding and enable efficient folding in the cellular environment. Trigger factor is a bacterial chaperone that binds to ribosomes, interacting with nascent polypeptides emerging from the ribosome and guiding their early folding steps. In contrast to the central role of the chaperone in promoting folding of newly synthesized proteins, its dynamic interactions with nascent chains emerging from the ribosome remain poorly understood. Here, we use single-molecule fluorescence and optical tweezers approaches to directly observe and characterize trigger factor interactions with a ribosome-bound client protein at increasing chain lengths. We find that trigger factor binding to nascent proteins is best described by a combination of multiple weak, dynamic interactions that are established after the chaperone docks onto the ribosome and evolve during polypeptide elongation. Application of mechanical force perturbs trigger factor binding, supporting a multivalent interaction model. This binding mode may help to stabilize nascent proteins against misfolding while allowing them to dynamically sample conformational space in search of their native structures.

biophysics↗

bioRxiv2024

Stick-slip unfolding favors self-association of expanded HTT mRNA

In Huntingtons Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

biophysics↗

bioRxiv2024

SOCS domain targets ECM assembly in lung fibroblasts and experimental lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease defined by a progressive decline in lung function due to scarring and accumulation of extracellular matrix (ECM) proteins. The SOCS (Suppressor Of Cytokine Signaling) domain is a 40 amino acid conserved domain known to form a functional ubiquitin ligase complex targeting the Von Hippel Lindau (VHL) protein for proteasomal degradation. Here we show that the SOCS conserved domain operates as a molecular tool, to disrupt collagen and fibronectin fibrils in the ECM associated with fibrotic lung myofibroblasts. Our results demonstrate that fibroblasts differentiated using TGF{beta}, followed by transduction with the SOCS domain, exhibit significantly reduced levels of the contractile myofibroblast-marker, -SMA. Furthermore, in support of its role to retard differentiation, we find that lung fibroblasts expressing the SOCS domain present with significantly reduced levels of -SMA and fibrillar fibronectin after differentiation with TGF{beta}. We show that adenoviral delivery of the SOCS domain in the fibrotic phase of experimental lung fibrosis in mice, significantly reduces collagen accumulation in disease lungs. These data underscore a novel function for the SOCS domain and its potential in ameliorating pathologic matrix deposition in lung fibroblasts and experimental lung fibrosis.

cell biology↗

bioRxiv2020

Multicohort Analysis of Publicly-available Monocyte Expression Data Identifies Gene Signatures to Accurately Monitor Subset-specific Changes in Human Diseases

Monocytes and monocyte-derived cells play important roles in the regulation of inflammation, both as precursors as well as effector cells. Monocytes are heterogeneous and characterized by three distinct subsets in humans. Classical and non-classical monocytes represent the most abundant subsets, each carrying out distinct biological functions. Consequently, altered frequencies of different subsets have been associated with inflammatory conditions, such as infections and autoimmune disorders including lupus, rheumatoid arthritis, inflammatory bowel disease, and, more recently, COVID-19. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and, consequently, poor reproducibility. Public transcriptomes provide an alternative source of data characterized by high statistical power and real world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in the levels of specific cell types. Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures, although derived using only datasets profiling healthy individuals, maintain their accuracy independent of the disease state in an independent cohort profiled by RNA-sequencing (AUC = 1.0). Furthermore, we demonstrate that our signatures are specific to monocyte subsets compared to other immune cells such as B, T, dendritic cells (DCs) and natural killer (NK) cells (AUC = 0.87~0.88, p<2.2e-16). This increased specificity results in estimated monocyte subset levels that are strongly correlated with cytometry-based quantification of cellular subsets (r = 0.69, p = 6.7e-4). Consequently, we show that these monocyte subset-specific signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.

systems biology↗

bioRxiv2020

Synthesis runs counter to the directional folding pathway of a nascent protein domain

Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

biophysics↗