Rachita Dash

J Biol Chem. 2026 Mar;302(3):111206. doi: 10.1016/j.jbc.2026.111206. Epub 2026 Jan 23.

ABSTRACT

The cancer glycocalyx is characterized by the overexpression of large glycoconjugates, including mucins, proteoglycans, and polysialic acid, which collectively increase glycocalyx thickness and stiffness to promote tumor survival, metastasis, and progression. Previous work demonstrated that enzymatic degradation of cancer cell-surface mucins reduced tumor burden and metastasis in mouse models of breast cancer, thus validating glycocalyx remodeling as a therapeutic strategy. However, this work relied on an engineered bacterial mucin-selective protease, or mucinase, which does not degrade other bulky glycoconjugates and raises immunogenicity concerns because of its bacterial origin. A human enzyme that can degrade cell-surface mucins and other bulky glycoconjugates would address both limitations. We screened all 15 human cathepsins for their ability to degrade purified and cell-surface mucins, since multiple cathepsins have previously been shown to degrade mucins within regions of dense glycosylation. We found that cathepsin K (CTSK) uniquely degrades cell-surface mucins, proteoglycans, and polysialylated glycoproteins, and we demonstrated that CTSK reduces total glycocalyx thickness. These findings establish CTSK as a promising starting point for the development of a glycocalyx-debulking enzyme for cancer therapeutics.

PMID:41581870 | PMC:PMC12969431 | DOI:10.1016/j.jbc.2026.111206

bioRxiv [Preprint]. 2025 Dec 7:2025.12.04.691947. doi: 10.64898/2025.12.04.691947.

ABSTRACT

It has been hypothesized that while random sequences are unlikely to fold into proteins of the length of globular proteins, repeated random sequences are more likely to adopt stably folded structures, with implications for molecular evolution. We used structure prediction methods to determine the foldability of approximately 120-residue sequences composed of 5-to 60-residue random repeats. With repeats of less than 30-residues, sequences were frequently discovered (1-12%) that fold with high confidence. For less than 60-residue repeats, we frequently observe β-solenoids, similar to those seen in natural proteins. We observe solenoids stabilized by apolar packing as well as ones stabilized by polar interactions with Ca ²⁺ in the core of the structure as in natural RTX domains. Helical bundles were observed with high frequency when insertions or deletions (INDELs) were included between blocks of repeating sequences. We also observed a new super-secondary structure consisting of a tightly wound α-helical screw, and experimentally confirmed its stability and structure by CD spectroscopy and X-ray crystallography. Thus, structure predictors can discover structures that are well out of the distribution of the data upon which they were trained. Beyond 40-residue repeat lengths very few sequences were predicted to fold. The small number of structures we observed were representative of well-established major classes of tertiary structures; greater sampling would be needed to discover novel structures from a random distribution. These studies illuminate dark matter regions of protein structure space and support previous predictions that proteins evolved through the assortment of shorter peptide sequences.

SIGNIFICANCE STATEMENT: The availability of powerful and accurate programs for predicting protein three-dimensional structures enables one to ask fundamental questions concerning the origin of folded functional proteins during evolution. We show that 120-residue proteins composed of random sequences repeated in tandem are predicted to be much more likely to fold than fully random proteins. These studies validate previous predictions that proteins evolved through the repetition and assortment of short peptide sequences. Also, some of the predicted structures represent novel conformations, which were confirmed experimentally. These findings advance our understanding of molecular evolution and have implications for design of novel proteins.

PMID:41573852 | PMC:PMC12822642 | DOI:10.64898/2025.12.04.691947

J Am Chem Soc. 2026 Jan 22. doi: 10.1021/jacs.5c13581. Online ahead of print.

ABSTRACT

Disulfide linkages of proteins are attractive yet underutilized sites for bioconjugation. Their use is hampered by limitations in existing disulfide bioconjugation chemistry including slow reaction kinetics, moderate yields, and suboptimal conjugate stability. Moreover, an intrinsic limitation of disulfide bioconjugation is that it can be performed only on proteins that either contain native cystine disulfide linkages or have been engineered by introducing two non-native cysteines that form this linkage during protein folding. Introducing two exogenous cysteines into a protein is not ideal as it renders it susceptible to misfolding. Herein, we report the StapleAld technology and combine it with the LAP tag technology (LAP-StapleAld) to overcome these limitations. StapleAld employs 2-formylallyl carboxylates for the rapid and quantitative formation of stable disulfide rebridged adducts appended with aldehyde tags for late-stage labeling. LAP-StapleAld integrates StapleAld with a chemoenzymatic approach to install lipoyl disulfide linkages at any desired site on any protein post-folding circumventing the protein misfolding problem, rendering disulfide bioconjugation universally applicable and location agnostic. Building on model studies on small molecules, we employ these platforms for the rapid, quantitative, and stable labeling of the cyclic peptides somatostatin and vasopressin and proteins including lysozyme, maltose-binding protein, eGFP, BtuF, and the therapeutic antibody, trastuzumab.

PMID:41572499 | DOI:10.1021/jacs.5c13581

Nat Commun. 2026 Jan 20;17(1):759. doi: 10.1038/s41467-026-68605-y.

ABSTRACT

Despite recent advances in targeted drug delivery, approved Antibody-Drug-Conjugates (ADCs) are still limited by the delivery of a restricted set of payloads with limited modes of action (MOA). Versatile linkers, applicable to functional groups prevalent across diverse pharmacophores are needed to expand this space. We present phosphoramidate-based self-immolative linker-units that facilitate stable attachment in serum and traceless drug release in the target cell of aliphatic and aromatic alcohols. Studies with camptothecins show that stability and release are tunable and that various intracellular trigger events can be exploited to ensure traceless drug delivery. Superior stability, in vivo efficacy, and pharmacokinetics (PK) compared to approved camptothecin ADCs are demonstrated. Moreover, we report targeted delivery of 10 different hydroxy-containing cytotoxins with different intracellular MOAs. In vivo studies with gemcitabine show excellent PK and efficacy, unlocking gemcitabine's full potential and illustrating the ability of the phosphoramidate-based linker system to expand the payload space for ADCs.

PMID:41554755 | PMC:PMC12820277 | DOI:10.1038/s41467-026-68605-y

ACS Appl Mater Interfaces. 2026 Jan 28;18(3):4805-4817. doi: 10.1021/acsami.5c20298. Epub 2026 Jan 19.

ABSTRACT

Polyacrylamide (PAM) hydrogels are extensively used as extracellular matrix mimics to study specific cell-material interactions. However, conventional biofunctionalization strategies lack chemo-selectivity and control over ligand density, compromising reproducibility and experimental reliability. In this work, we introduce firefly luciferin-inspired click ligation to enable efficient and tunable biofunctionalization of PAM hydrogels. A novel acrylamide-based comonomer containing cyanobenzothiazole (CBT) moieties is synthesized and incorporated into PAM hydrogels. CBT mediates biofunctionalization of PAM with N-Cys bearing biomolecules via luciferin click chemistry. Biofunctionalization occurs within only a few minutes, under mild conditions, with high efficiency, not requiring light exposure. Compared to the widely used sulfo-SANPAH (SS)-based approach, our method offers enhanced biofunctionalization efficiency, homogeneity, and control over biomolecule loading while preserving biochemical functionality. This translates into improved presentation of cell-adhesive cues, resulting in significantly increased cell attachment, spreading, and proliferation, as demonstrated by using label-free holotomography. The novel luciferin click ligation offers a robust, efficient, and reproducible alternative for PAM biofunctionalization, providing precise control over the ligand density while maintaining bioactivity. As PAM hydrogels continue to evolve into increasingly sophisticated mechanobiology tools, our approach may serve as a new standard for engineering the interfacial properties of these materials to achieve robust two-dimensional (2D) cell culture platforms for fundamental studies in cell-material interactions.

PMID:41549978 | DOI:10.1021/acsami.5c20298

J Am Chem Soc. 2026 Jan 28;148(3):2890-2895. doi: 10.1021/jacs.5c19988. Epub 2026 Jan 16.

ABSTRACT

Thiopeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that show promise for drug discovery. Their biosynthesis depends on leader peptide recognition, whereby multiple enzymes are recruited to process a core region into the mature natural product. Here, we identify sequence determinants of the leader peptide-enzyme dynamics in a thiopeptide biosynthesis. Using the flexible in vitro (FIT)-Laz translation platform, we examined how leader mutations influence modifications performed by the enzymes that together complete lactazole biosynthesis. Initial DNA template translations informed single amino acid saturation mutagenesis using mRNA display. This revealed leader mutations that modulate enzyme recognition, as validated by aligning enrichment scores with observed modification. This study provides important insights into the underexplored role of RiPP leader peptides and informs the design of improved pseudonatural product libraries.

PMID:41542783 | DOI:10.1021/jacs.5c19988

Proc Natl Acad Sci U S A. 2026 Jan 20;123(3):e2516051123. doi: 10.1073/pnas.2516051123. Epub 2026 Jan 16.

ABSTRACT

Gasdermin D (GSDMD) is the principal executor of pyroptosis, a form of proinflammatory programmed cell death misregulation of which is associated with numerous diseases. Despite significant interest, no specific GSDMD inhibitors have been developed for clinical use so far. Here, we developed a strategy to generate mRNA-displayed libraries of bicyclic cysteine-rich peptides (bCRP), and utilized these libraries to develop potent peptide ligands to full-length GSDMD using a two-stage discovery process. Initial hit compounds were de novo discovered from GSDMD affinity selections using Random Nonstandard Peptides Integrated Discovery system, and were then optimized using mRNA display-based saturation mutagenesis. The resulting bCRPs bound to full-length GSDMD (best K_D = 125 nM) and stabilized it against cleavage by caspase proteases. The bCRPs prevented the pore formation in liposome leakage assays and inhibited the secretion of IL-1β and lactate dehydrogenase from pyroptotic THP-1 cells. The potency, high metabolic stability, and synthetic accessibility of the discovered compounds make them promising leads for the development of GSDMD-targeting therapeutics.

PMID:41543906 | PMC:PMC12818410 | DOI:10.1073/pnas.2516051123

Proc Natl Acad Sci U S A. 2026 Jan 6;123(1):e2505932123. doi: 10.1073/pnas.2505932123. Epub 2026 Jan 2.

ABSTRACT

The precise recognition of specific peptide-major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in E. coli of H-2D^b and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

PMID:41481462 | PMC:PMC12773744 | DOI:10.1073/pnas.2505932123

Curr Opin Chem Biol. 2026 Feb;90:102641. doi: 10.1016/j.cbpa.2025.102641. Epub 2025 Dec 24.

NO ABSTRACT

PMID:41447833 | DOI:10.1016/j.cbpa.2025.102641

Angew Chem Int Ed Engl. 2026 Feb 9;65(7):e19027. doi: 10.1002/anie.202519027. Epub 2025 Dec 29.

ABSTRACT

Nonviral protein vehicles for RNA delivery hold significant promises for various biotechnological applications due to their scalability, ease of engineering, and high biocompatibility. Directed evolution is a powerful approach to enhance protein activity, yet few nonviral protein vehicles have been evolved due to the challenges in linking genotype and phenotype. Here, we report engineering and directed evolution of a neo-nucleocapsid enabling receptor-specific RNA delivery. We applied the DogTag/DogCatcher protein ligation system to an artificial nucleocapsid and conducted directed evolution to obtain nucleocapsid displaying proteins. To create designer neo-nucleocapsids, we lasso-grafted a pharmacophore sequence of a pseudo-cyclic peptide binding to the hepatocyte growth factor receptor MET into the loop of DogCatcher. After the cellular-based directed evolution, endocytic uptake of neo-nucleocapsids was improved whereas cytosolic release and subsequent protein expression from the delivered RNA were not detected. This experimental approach provides a versatile system for constructing protein cages delivering various cargos to specific cells.

PMID:41460065 | PMC:PMC12887611 | DOI:10.1002/anie.202519027