Biology subjects

Pokornowska, M.

Publications and source records attributed to Pokornowska, M..

3 recordsLinked to original sources

bioRxiv2026

Multi-Step Hsp70 Recruitment by Class B J-Domain Proteins Drives Efficient Protein Disaggregation

Protein disaggregation by the Hsp70 system is essential for maintaining cellular proteostasis, especially under stress and proteotoxic diseases. J-Domain Proteins (JDPs) assist and regulate Hsp70 by stimulating substrate binding and ATP hydrolysis through their signature J-domain. In Class B JDPs, which play a key role in disaggregation of amyloids and amorphous aggregates, the J-domain is autoinhibited and requires activation via an auxiliary interaction with the EEVD motif of Hsp70. We reconstituted Hsp70 system from yeast and human to examine how the distinctive properties of Class B JDPs--autoinhibition and EEVD binding--promote the accumulation of Hsp70 molecules at the aggregate surface, facilitating aggregate disassembly. We revise the Hsp70 functional cycle to account for EEVD binding, showing that it enables the formation of a JDP-Hsp70-aggregate complex. This implies a model of multi-step Hsp70 clustering on aggregates, in which Class B JDPs in complex with aggregate-bound Hsp70 recruit additional Hsp70 molecules through an EEVD-independent mechanism. Experiments with the Hsp70 variant lacking EEVD validate this model but also reveal negative consequences of the stable Hsp70-JDP interaction that make the J-domain autoinhibition indispensable. Collectively, our results highlight EEVD binding and JDP autoinhibition as a coupled regulatory strategy leading to enhanced Hsp70-mediated disaggregation.

biochemistry↗

bioRxiv2023

Balanced Interplay Between Hsp110, Hsp70 and Class B J-Domain Protein Improves Aggregate Disassembly

Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing and disease. Hsp70 solubilizes aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and Nucleotide Exchange Factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with Class B than Class A JDPs and requires the auxiliary interaction between Class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the substoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.

biochemistry↗

bioRxiv2020

RNA and DNA G-quadruplexes bind to human Dicer and inhibit its activity

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

biochemistry↗