Biology subjects

Kriwacki, R. W.

Publications and source records attributed to Kriwacki, R. W..

7 recordsLinked to original sources

bioRxiv2025

Protein Dynamics at Different Timescales Unlock Access to Hidden Post-Translational Modification Sites

Post-translational modifications (PTMs) alter the proteome in response to intra- and extracellular signals, providing fundamental information processing in development, homeostasis and disease1-5. Here, we used proteome-wide structure predictions to investigate the structural context of protein modification sites and found nearly a fifth to be buried within proteins6. These cryptic sites within folded domains showed evolutionary conservation across species, as well as significantly less variability within the human population. We also found significantly more modification sites to be affected by recurrent cancer mutations and disease variants, with some variants appearing to mimic the modified or unmodified state, supporting their functionality and disease relevance. To understand how these buried sites become modified, we studied 23 phosphorylation sites across seven proteins using all-atom molecular dynamics simulations. The simulations revealed a range of mechanisms spanning broad timescales, ranging from picosecond fluctuations to seconds for proline isomerisation-gated phosphorylation sites. Notably, some sites remained fully buried in the native structure. Some conformational states with exposed cryptic sites resembled structures of co-translational protein folding intermediates formed on the ribosome7, indicating an interplay between protein biogenesis, folding, modifications, and signalling. We propose more broadly that protein conformational fluctuations may serve as structure-specific regulatory mechanisms by kinetically burying or exposing the unmodified, and in some cases even modified, sidechains, thereby expanding the functional versatility of proteins and enabling signal transduction across diverse timescales.

bioinformatics↗

bioRxiv2025

Proteome-wide computational analyses reveal links between protein condensate formation and RNA biology

Biomolecular condensates mediate dynamic compartmentalization of cellular processes. The multivalent interactions that underlie biomolecular condensation are often promoted by intrinsically disordered regions (IDRs) within proteins. While the role of IDRs in biomolecular condensates is well appreciated, predicting whether an IDR forms condensates in cells remains challenging. Here, we developed a machine learning model to predict condensation behavior of IDRs, analyzing 215 IDRs from fusion oncoproteins in HEK293T cells. Our study identified distinct sequence-derived physicochemical features associated with condensation. Leveraging these data, our model predicts that [~]12% of the [~]13,000 IDRs in the human proteome are likely to form cellular condensates. Proteins with condensate-forming IDRs are enriched in functions involving RNA-related processes and membrane-less organelles (MLOs), highlighting their role in MLO assembly and function. Our model, available via the SAK3.0 web server (https://sak.stjude.org), provides a powerful resource for studying IDR-driven phase separation across proteomes, offering insights into biomolecular condensates and their biological roles.

bioinformatics↗

bioRxiv2025

Granular component sub-phases direct ribosome biogenesis in the nucleolus

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GCs spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GCs spatial heterogeneity. SURF6s association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates--suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

biochemistry↗

bioRxiv2024

A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

Mitochondria maintain a biochemical environment that cooperates with BH3-only proteins (e.g., BIM) to potentiate BAX activation, the key event to initiate physiological and pharmacological forms of apoptosis. The sphingosine-1-phosphate metabolite 2-trans-hexadecenal (2t-hexadecenal) is one such component described to support BAX activation, but molecular mechanisms remain largely unknown. Here, we utilize complementary biochemical and biophysical techniques to reveal that 2t-hexadecenal non-covalently interacts with BAX, and cooperates with BIM to stimulate early-activation steps of monomeric BAX. Integrated structural and computational approaches reveal 2t-hexadecenal binds an undefined region - a hydrophobic cavity formed by core-facing residues of 5, 6, and gated by 8 - we now term the "BAX actuating funnel" (BAF). We define alkenal length and 8 mobility as critical determinants for 2t-hexadecenal synergy with BIM and BAX, and demonstrate that proline 168 allosterically regulates BAF function. Collectively, this work imparts detailed molecular insights advancing our fundamental knowledge of BAX regulation and identifies a regulatory region with implications for biological and therapeutic opportunities.

cell biology↗

bioRxiv2024

MOZ and HBO1 Histone Acetyltransferase Complexes Are Molecular Dependencies and Therapeutic Targets in NUP98-Rearranged Acute Myeloid Leukemia

NUP98 fusion oncoproteins (FOs) are a hallmark of childhood acute myeloid leukemia (AML) and drive leukemogenesis through liquid-liquid phase separation-mediated nuclear condensate formation. However, the composition and consequences of NUP98 FO-associated condensates are incompletely understood. Here we show that MYST family histone acetyltransferase (HAT) complex proteins including MOZ/KAT6A, HBO1/KAT7, and the common MOZ/HBO1 complex subunit BRPF1 associate with NUP98 FOs on chromatin and within condensates. MYST HATs are molecular dependencies in NUP98-rearranged (NUP98-r) leukemia, and genetic inactivation or pharmacologic inhibition of Moz and Hbo1 impairs NUP98-r cell fitness. MOZ/HBO1 inhibition decreased global H3K23ac levels, displaced NUP98::HOXA9 from chromatin at the Meis1 locus, and led to myeloid cell differentiation. Additionally, MOZ/HBO1 inhibition decreased leukemic burden in multiple NUP98-r leukemia xenograft mouse models, synergized with Menin inhibitor treatment, and was efficacious in Menin inhibitor-resistant cells. In summary, we show that MYST family HATs are therapeutically actionable dependencies in NUP98-r AML. SIGNIFICANCE STATEMENTMOZ and HBO1 associate with NUP98 fusion oncoprotein condensates to drive leukemogenesis. Inhibition of their histone acetyltransferase activity is an effective therapeutic strategy in NUP98-rearranged leukemias, including those resistant to Menin inhibition. Moreover, combined MOZ/HBO1 and Menin inhibition is synergistic, supporting clinical translation to improve outcomes of NUP98 FO-driven leukemias.

cancer biology↗

bioRxiv2023

Biomolecular condensates form spatially inhomogeneous network fluids

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

biophysics↗

bioRxiv2019

Composition dependent phase separation underlies directional flux through the nucleolus

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

biophysics↗