Biology subjects

Banik, S. M.

Publications and source records attributed to Banik, S. M..

4 recordsLinked to original sources

bioRxiv2026

Ratiometric transcriptional activation by protein degradation

Cells can respond to alterations in the abundances of specific proteins through transcriptional outputs. Synthetic approaches inspired by native post-transcriptional circuits that convert protein abundance changes into programmable gene expression would be transformative. Here, we discover and describe design principles that effectively convert protein degradation into transcriptional outputs in live cells. We define ratiometric transcriptional activation, where control over the ratio between a transcriptional inhibitor-protein of interest fusion and transcription factor enables detection of abundance changes with high sensitivity at scale. We show that ratiometric transcriptional activation can be implemented in single cells using triply orthogonal circuits or in multicellular pools, operating independently of mechanism of protein downregulation and enabling simultaneous detection of multiple protein downregulation events through outputs such as cell survival, fluorescent protein expression, or barcode sequencing. These circuits can be applied to oncogenic targets and enable discovery of new molecular glue degraders.

synthetic biology↗

bioRxiv2026

Programmed Manipulation of RNA Targets By Human Argonaute 2

Nucleic acid manipulation using programmable ribonucleoprotein complexes (RNPs) has enabled transformative research tools and led to new therapeutic strategies. RNA directly regulates diverse cellular processes,1 is a crucial mediator of protein synthesis, and offers advantages in therapeutic targeting and fundamental discovery complementary to those of DNA.2 ISC-3 and Cas-based4,5 scaffolds where the RNP is fused to an effector protein can alter RNA sequence, structure, and function. However, the non-human origins underlying these systems create challenges in therapeutic translation and the presence of non-native proteins can have unintended and little understood effects on cells.6-8 Systematic repurposing of human proteins, which have been optimized in the cellular environment by evolution, for expanded programmable functions could reveal new biological principles and bypass the limitations of foreign proteins. Here, we demonstrate that the catalytic engine of the RNA-interference (RNAi) pathway, human Argonaute 2 (AGO2),9,10 can be repurposed as a modular targeting domain, and when fused to a C-to-U deaminase, enable AGO-Led Targeted Editing of RNA (ALTER). Using guide RNAs which remodel target RNA structure for selective editing and reduced nuclease activity, we show that ALTER can act on a variety of target transcripts including endogenous mRNAs and lncRNAs, with activities comparable or exceeding those of Cas-based systems.11,12 Despite its human origin and role in RNAi, transcriptome-wide RNAseq revealed lower levels of off-target editing compared to Cas-based editing systems. These results demonstrate that AGO2 can be rationally redirected from RNAi to a broader spectrum of RNA manipulations, establishing that intact human proteins can be reconfigured for expanded molecular function.

biochemistry↗

bioRxiv2024

A secondary β-hydroxybutyrate metabolic pathway linked to energy balance

{beta}-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.

biochemistry↗

bioRxiv2023

Targeted Protein Relocalization via Protein Transport Coupling

Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2-4. The localization of numerous proteins has been annotated5-7, and pharmacologically relevant approaches for precise rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules which harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could provide an avenue for targeted protein relocalization for interactome-rewiring therapeutics. To realize this concept, we deploy a quantitative approach to identify features which govern the ability to hijack protein trafficking, develop a collection of shuttle proteins and ligands, and demonstrate relocalization of proteins bearing endogenous localization signals. Using a custom imaging analysis pipeline, we show that endogenous localization signals can be overcome through molecular coupling of target proteins to shuttle proteins containing sufficiently strong native localization sequences expressed in the necessary abundance. We develop nuclear hormone receptors as viable shuttles which can be harnessed with Targeted Relocalization Activating Molecules (TRAMs) to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43 {Delta}NLS, and FUSR495X. Small molecule-mediated relocalization of FUSR495X to the nucleus from the cytoplasm reduced the number of cellular stress granules in a model of cellular stress. Using Cas9-mediated knock-in tagging, we demonstrate nuclear enrichment of both low abundance (FOXO3a) and high abundance (FKBP12) endogenous proteins via molecular coupling to nuclear hormone receptor trafficking. Finally, small molecule-mediated redistribution of NMNAT1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the WldS gain-of-function phenotype from mice resistant to certain types of neurodegeneration8. The concept of targeted protein relocalization could therefore nucleate approaches for treating disease through interactome rewiring.

biochemistry↗