Delivery of Peptide Coacervates to Form Stable Interaction Hubs in Cells.

Cells contain membrane-bound and membraneless organelles that operate as spatially distinct biochemical niches. However, these subcellular reaction centers lose fidelity with aging and as a result of disease. A grand challenge for biomedicine is restoring or augmenting cellular functionalities.

Conditional Localization Pharmacology Manipulates the Cell Cycle with Spatiotemporal Precision.

Traditional pharmacology has limited control of drug activity and localization in space and time. Herein, we described an approach for kinase regulation using conditional localization pharmacology (CLP), where an inactive caged inhibitor is localized to a site of interest in a dormant state using intracellular protein tethering. The activity of the inhibitor can be regulated with spatial and temporal precision in a live cellular environment using light.

The central spindle drives anaphase chromosome segregation.

Anaphase chromosome segregation depends on forces exerted by spindle microtubules. In the current model, forces on chromosomes are mediated through the spindle poles: sliding of antiparallel microtubules in the central spindle pushes poles apart, while kinetochore microtubule (kMT) depolymerization pulls chromosomes towards the poles. Here we show that the central spindle is directly linked to the chromosomes rather than the poles in anaphase, based on three lines of evidence.

"Off-pore" nucleoporins relocalize heterochromatic breaks through phase separation.

Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and "safe" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear.

SUMO promotes DNA repair protein collaboration to support alternative telomere lengthening in the absence of PML.

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT.

Duo-Chol: A Photoconvertible Live Cell Imaging Tool for Tracking Cholesterol.

Investigating cholesterol trafficking pathways continues to be of significant scientific interest owing to its homeostasis being associated with several debilitating cardiovascular and neurodegenerative diseases including atherosclerosis, Niemann-Pick's disease, Alzheimer's disease, and Parkinson's disease. To further our understanding of cholesterol trafficking, it is imperative to develop new fluorescent probes that possess improved photostability, low efflux, and high spatial and temporal resolution for live-cell imaging. In this study, we developed a photoconvertible fluorescent cholesterol analog, Duo-Chol, enabling the improved spatiotemporal fluorescence imaging of the dynamic localization of cholesterol in live cells.

Interplay of condensate material properties and chromatin heterogeneity governs nuclear condensate ripening.

Nuclear condensates play many important roles in chromatin functions, but how cells regulate their nucleation and growth within the complex nuclear environment is not well understood. Here, we report how condensate properties and chromatin mechanics dictate condensate growth dynamics in the nucleus. We induced condensates with distinct properties using different proteins in human cell nuclei and monitored their growth.

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML.

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity.

Solid-Phase Photochemical Peptide Homologation Cyclization.

Forging new C(sp)-C(sp) bonds to central positions within a peptide backbone is critical for the development of new therapeutics and chemical probes. Currently, there are no methods for decarboxylating Asp and Glu side chains solid-phase photochemically or using such radicals to form peptide macrocycles. Herein, electron-donor-acceptor complexes between Hantzsch ester and on-resin peptide -hydroxyphthalimide radical precursors are used to access these radicals, demonstrated with two-carbon homologations and homologation cyclizations of Atosiban and RGDf.

A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation.

To mimic the levels of spatiotemporal control that exist in nature, tools for chemically induced dimerization (CID) are employed to manipulate protein-protein interactions. Although linker composition is known to influence speed and efficiency of heterobifunctional compounds, modeling or in vitro experiments are often insufficient to predict optimal linker structure. This can be attributed to the complexity of ternary complex formation and the overlapping factors that impact the effective concentration of probe within the cell, such as efflux and passive permeability.

Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination.

Genetic diseases are often caused by nonsense mutations, but only one TRID (translation readthrough inducing drug), ataluren, has been approved for clinical use. Ataluren inhibits release factor complex (RFC) termination activity, while not affecting productive binding of near-cognate ternary complex (TC, aa-tRNA.eEF1A.

CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells.

Solid-Phase Photochemical Decarboxylative Hydroalkylation of Peptides.

The compatibility of photochemistry with solid-phase peptide synthesis is demonstrated via photochemical hydroalkylation to form C(sp)-C(sp) bonds between on-resin Giese acceptors and redox-active esters. Both iridium-based photocatalysts and Hantszch ester led to high yields, with final reaction conditions producing full conversions within 30 min under ambient conditions. The chemistry is compatible with a broad range of peptide side chains, redox-active esters, and resin.

Rules for the design of aza-glycine stabilized triple-helical collagen peptides.

The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen's repeating (Glycine-Xaa-Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position.

Tension promotes kinetochore-microtubule release by Aurora B kinase.

To ensure accurate chromosome segregation, interactions between kinetochores and microtubules are regulated by a combination of mechanics and biochemistry. Tension provides a signal to discriminate attachment errors from bi-oriented kinetochores with sisters correctly attached to opposite spindle poles. Biochemically, Aurora B kinase phosphorylates kinetochores to destabilize interactions with microtubules.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E.

Multivalent, Soluble Nano-Self Peptides Increase Phagocytosis of Antibody-Opsonized Targets while Suppressing "Self" Signaling.

Macrophages engulf "foreign" cells and particles, but phagocytosis of healthy cells and cancer cells is inhibited by expression of the ubiquitous membrane protein CD47 which binds SIRPα on macrophages to signal "self". Motivated by some clinical efficacy of anti-CD47 against liquid tumors and based on past studies of CD47-derived polypeptides on particles that inhibited phagocytosis of the particles, here we design soluble, multivalent peptides to bind and block SIRPα. Bivalent and tetravalent nano-Self peptides prove more potent ( ∼ 10 nM) than monovalent 8-mers as agonists for phagocytosis of antibody opsonized cells, including cancer cells.

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions.

RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling.

Rational design of small molecule fluorescent probes for biological applications.

Fluorescent small molecules are powerful tools for visualizing biological events, embodying an essential facet of chemical biology. Since the discovery of the first organic fluorophore, quinine, in 1845, both synthetic and theoretical efforts have endeavored to "modulate" fluorescent compounds. An advantage of synthetic dyes is the ability to employ modern organic chemistry strategies to tailor chemical structures and thereby rationally tune photophysical properties and functionality of the fluorophore.

Nuclear body phase separation drives telomere clustering in ALT cancer cells.

Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically induced dimerization approach to induce de novo APB condensation in live cells without DNA damage.