Biology subjects

Chakrabarti, J.

Publications and source records attributed to Chakrabarti, J..

5 recordsLinked to original sources

bioRxiv2022

Genetically Engineered Human Pituitary Corticotroph Tumor Organoids Exhibit Divergent Responses To Glucocorticoid Receptor Modulators

Cushings disease (CD) is a serious endocrine disorder attributed to an ACTH-secreting pituitary neuroendocrine tumor (PitNET) that subsequently causes chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced the predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD patients. HIGHLIGHTSO_LICushing disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that leads to chronic hypercortisolemia C_LIO_LIMifepristone (Korlym(R)), a non-selective glucocorticoid receptor (GR) antagonist, is an approved treatment for patients with Cushing disease, and competes with the binding of cortisol to the GR as well as the binding of progesterone to the progesterone receptor. C_LIO_LIRelacorilant is an investigational selective GR modulator in development for the treatment of Cushing syndrome that, unlike mifepristone, does not bind to the other hormone receptors. C_LIO_LIUnlike mifepristone, relacorilant does not significantly raise systemic cortisol levels, and cases of PitNET regression with relacorilant have been reported. However, the mechanisms behind these clinical differences remained unknown. C_LIO_LIPitNET organoids were generated from: 1) CRISPR-Cas9 gene editing of patient iPSCs, and 2) CD patient corticotroph PitNETs (hPITOs) and used to compare the diverse effects of mifepristone and relacorilant in a human-relevant model that recapitulates the PitNET microenvironment in vitro. C_LIO_LIMifepristone and relacorilant have different effects on the induction of somatostatin receptor (SSTR) SSTR2 and SSTR5 expression, ACTH secretion and PitNET organoid proliferation and apoptosis. C_LI BRIEF COMMENTARYO_ST_ABSBackgroundC_ST_ABSCushings disease (CD), a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) leads to chronic hypercortisolemia. Approved for the treatment for CD, Mifepristone (Korlym(R)) is a non-selective glucocorticoid receptor (GR) antagonist with additional competitive binding with progesterone for the progesterone receptor. Relacorilant, an investigational selective GR modulator in development for the treatment of CD, does not bind to the other hormone receptors. Translational SignificancePatient-derived PitNET organoids recapitulate the tumor microenvironment in vitro. PitNET organoids revealed the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD.

physiology↗

bioRxiv2022

Conformational fluctuations in molten globule state of α-lactalbumin

Molten globule (MG) state is an intermediate state of protein observed during folding into native structure. MG state of protein is induced by various denaturing agent (like Urea), extreme pH, pressure and heat. Experiments suggest that MG state of some protein is functionally relevant even if there is no well-defined tertiary structure. Earlier experimental and theoretical studies suggest that MG state of the protein is dynamic in nature, where conformational states are interconverted in nanosecond time scales. These observations lead us to study and compare conformational fluctuations of MG state to those of intrinsic disordered protein (IDP). We consider -Lactalbumin(aLA) protein, which shows MG state at low pH upon removal of calcium (Ca2+) ion. We use constant pH molecular dynamics simulation (CpHMD) to maintain low pH during simulation. We use the dihedral principal component analysis, the density based clustering method and the machine learning technique to identify the conformational fluctuations. We observe metastable states in the MG state. The residues containing the essential coordinates responsible for metastability belong to stable helix in crystal structure, but most of them prefer unstructured or bend conformation in MG state. These residues control the exposure of the putative binding residues for fatty acids. Thus, the MG state of protein behaves as intrinsic disorder protein, although the disorder here is induced by external conditions.

biophysics↗

bioRxiv2022

Effect on the conformations of spike protein of SARS-CoV-2 due to mutation

The spike protein of SARS CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations appeared in variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wildtype from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between N-terminal domain and receptor binding domain leading to an obtuse angle cosine{theta} distribution in the trimeric structure in spike protein. Thus, increase in open-state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming linker loop where many residues undergo stabilization in the open state compared to the closed one.

biophysics↗

bioRxiv2022

Live-Cell Imaging in Human Colonic Monolayers Reveals Erk Waves Limit the Stem Cell Compartment to Maintain Epithelial Homeostasis

The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here we developed a 2D patient derived organoid (PDO) screening platform to study tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If Erk is activated acutely across all cells instead of in wavelike patterns, then tissue patterning, and stem cell maintenance are lost. Conversely, if Erk activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated Erk signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how Erk can antagonize stem cells yet support cell replacement and the function of the gut.

cell biology↗

bioRxiv2021

Immune escape facilitation by mutations of epitope residues in RdRp of SARS-CoV-2

SARS-CoV-2 has considerably higher mutation rate. SARS-CoV-2 possesses a RNA dependent RNA polymerase (RdRp) which helps to replicate its genome. The mutation P323L in RdRp is associated with the loss of a particular epitope (321-327) from this protein which may influence the pathogenesis of the concern SARS-CoV-2 through the development of antibody escape variants. We consider the effect of mutations in some of the epitope regions including the naturally occurring mutation P323L on the structure of the epitope and their interface with paratope using all-atom molecular dynamics (MD) simulation studies. P323L mutations cause conformational changes in the epitope region by opening up the region associated with increase in the radius of gyration and intramolecular hydrogen bonds, making the region less accessible. Moreover, the fluctuations in the dihedral angles in the epitope:paratope (IgG) interface increase which destabilize the interface. Such mutations may help in escaping antibody mediated immunity of the host.

bioinformatics↗