Biology subjects

Shalgi, R.

Publications and source records attributed to Shalgi, R..

8 recordsLinked to original sources

bioRxiv2026

Chaperone isoform and interactome mapping reveals functional diversification of DNAJA2-DNAJA4 complexes via stress-regulated isoforms

The human HSP70 chaperone network maintains cellular proteostasis through a diverse repertoire of HSP70s and co-chaperones. Here we examine alternative isoforms and co-chaperone hetero-complexes as additional sources of network complexity. To that end, we systematically mapped the isoform, tissue-expression, and interaction landscapes of the human HSP70 network, revealing a modular interactome containing known and novel DNAJ-DNAJ interactions. We found both tissue-expression isoform divergence as well as widespread alternative-canonical isoform co-expression, suggesting additional modes of functional diversification. Focusing on the uncharacterized DNAJA2-DNAJA4 hetero-complex, we identified the stress-inducible isoform DNAJA4-CTD-II. DNAJA4-CTD-II formed hetero-complexes with DNAJA2 and DNAJA4, with both interactions enhanced following sodium arsenite stress. Functionally, DNAJA4-CTD-II co-localized with TDP-43 aggregates and significantly suppressed their accumulation in a DNAJA2-dependent manner. Together, our data reveal extensive, uncharted isoform and interaction complexity within the HSP70 network, and uncover isoform-dependent hetero-complex remodeling as a new layer of chaperone network regulation.

systems biology↗

bioRxiv2024

Targeting FUS-ALS aggregation with Proteasome Inhibitors

ALS, Amyotrophic lateral sclerosis, a devastating neurodegenerative disease (ND) with no cure, is often caused by abnormal cytosolic aggregation of RNA-binding proteins, the most well-known of which are TDP-43 and FUS. The proteasome is considered one of the major systems that degrades misfolded, including ND-associated, proteins, thereby acting to reduce aggregation, while inhibition of the proteasome increases aggregation. Unexpectedly, we found that proteasome inhibitor treatment significantly reduced ALS-associated mutant FUS aggregation in cells and in primary neurons. This is in sharp contrast to most other ND-associated aggregating proteins, including Huntingtin and TDP-43, for which proteasome inhibitors enhanced aggregation. We further found that this inhibitory effect is dependent on the transcription factor HSF1, suggesting that the underlying mechanism of this effect is transcriptionally-mediated. Since heat shock treatment did not show any effect on FUS aggregation, we hypothesized that proteasome inhibitors elicit a transcriptional program distinct of that of heat shock, which is protective of FUS aggregation. We identified BAG3, a co-chaperone that cooperates with HSP70 in reducing FUS aggregation, as a significant mediator of this effect. We therefore propose BBB-permeable proteasome inhibitors as a potential therapy specific to ALS-FUS.

molecular biology↗

bioRxiv2023

Engineering mice for female-biased progeny without impacting genetic integrity and litter size

The ability to influence the sex ratio of mammalian offspring has applications in agriculture and animal welfare. Here we describe a genetic system in mice that produces predominantly female progeny, reaching approximately ninety percent, without affecting litter size or introducing genetic modifications into the offspring. The system consists of a doxycycline-regulated cassette inserted into the Y chromosome, encoding dCas9 and RNA guides intended to impair the function of Y bearing sperm. While the cassette was originally intended to repress a spermatid maturation gene, our analyses did not detect meaningful changes in gene expression or sperm function that would account for the phenotype. Nevertheless, male mice carrying the cassette consistently produced female biased litters, and the effect was reversed by doxycycline, confirming that it depends on cassette expression. Similar female bias was observed following in vitro fertilization using sperm from transgenic males. These findings demonstrate a reproducible, genetically confined, and conditionally regulated system for sex ratio bias in mice. The strategy may prove adaptable across species, even without a complete mechanistic understanding. Graphical AbstractA single line genetic system producing female biased, genetically unmodified progeny in mice by conditionally impairing Y bearing sperm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/568055v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@5f23b3org.highwire.dtl.DTLVardef@20ed2corg.highwire.dtl.DTLVardef@1b3007dorg.highwire.dtl.DTLVardef@e54cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryY chromosome manipulation yields mostly female mice, preserving litter size without genetic alteration in females. Significance statementThis study describes a genetic approach for producing predominantly female offspring in mice without altering litter size or transmitting genetic modifications to the progeny. A cassette inserted into the Y chromosome of males induces a strong female bias in offspring through a mechanism that remains unclear. The female offspring do not inherit the transgene and are genetically unmodified. This system provides a simple and conditional platform for sex ratio control in mammals, with potential applications in animal breeding and welfare.

bioengineering↗

bioRxiv2022

Stress-induced transcriptional readthrough into neighboring genes is linked to intron retention

Exposure to certain stresses leads to readthrough transcription. Here we found that readthrough transcription often proceeds into the proximal gene downstream, in a phenomenon termed "read-in". Using polyA-selected RNA-seq data from mouse fibroblasts, we identified widespread read-in in heat shock, oxidative and osmotic stress conditions. We found that read-in genes share distinctive genomic characteristics; they are extremely short, and highly GC rich. Furthermore, using ribosome footprint profiling we found that translation of read-in genes is significantly reduced. Strikingly, read-in genes show extremely high levels of intron retention during stress, mostly in their first intron, which is not explained by features usually associated with intron retention, such as short introns and high GC content. Finally, we found that first introns in read-in genes have weaker splice sites. Our data portray a relationship between read-in and intron retention, suggesting it may have co-evolved to facilitate reduced translation of read-in genes during stress.

genomics↗

bioRxiv2021

Differential roles for DNAJ isoforms in HTT-polyQ and mutant FUS aggregation modulation revealed by chaperone network screens

Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntingtons disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completely different than those of HTT-polyQ. Interestingly, different naturally-occurring isoforms of DNAJ chaperones had opposing effects on HTT-polyQ vs. mutFUS aggregation. We identified a complex of the full length (FL) DNAJB14 and DNAJB12 isoforms which substantially alleviated mutFUS aggregation, in an HSP70-dependent manner. Their naturally occurring short isoforms were unable to form the complex, nor to interact with HSP70, and lost their ability to reduce mutFUS aggregation. In contrast, the short isoform of DNAJB12 significantly alleviated HTT-polyQ aggregation, while DNAJB12-FL aggravated HTT-polyQ aggregation. Finally, we demonstrated that full-length DNAJB14 ameliorated mutFUS aggregation compared to DNAJB14-short in primary neurons. Together, our data unraveled distinct molecular properties required for aggregation protection in different neurodegenerative diseases, and revealed a new layer of complexity of the chaperone network elicited by naturally occurring J-protein isoforms, highlighting functional diversity among the DNAJ family.

molecular biology↗

bioRxiv2019

Fluorescent polysome profiling reveals stress-mediated regulation of HSPA14-ribosome interactions

Ribosome-associated factors play important roles in regulation of translation in response to various physiological and environmental signals. Here we present fluorescent polysome profiling, a new method that provides simultaneous detection of UV and fluorescence directly from polysome gradients. We demonstrate the capabilities of the method by following the polysome incorporation of different fluorescently tagged ribosomal proteins in human cells. Furthermore, we used fluorescent polysome profiling to examine chaperone-ribosome interactions, and characterized their changes in response to proteotoxic stress conditions. We revealed dynamic regulation of HSPA14-polysome association in response to heat shock, with a marked heat shock-mediated increase in the polysome-association of HSPA14. Our data suggest a model whereby HSPA14 dimerization is increased upon heat shock. We therefore established fluorescent polysome profiling as a streamlined method, that can enhance and facilitate the broad exploration of ribosome-associated factors and their regulation.

molecular biology↗

bioRxiv2019

Cellular proteostasis decline in human senescence

Proteostasis collapse, the diminished ability to maintain protein homeostasis, has been established as a hallmark of nematode aging. However, whether proteostasis collapse occurs in humans has remained unclear. Here we demonstrate that proteostasis decline is intrinsic to human senescence. Using transcriptome-wide characterization of gene expression, splicing and translation, we found a significant deterioration in the transcriptional activation of the heat shock response in stressed senescent cells. Furthermore, phosphorylated HSF1 nuclear localization and distribution were impaired in senescence. Interestingly, alternative splicing regulation was also dampened. Surprisingly, we found a decoupling between different Unfolded Protein Response (UPR) branches in stressed senescent cells. While young cells initiated UPR-related translational and transcriptional regulatory responses, senescent cells showed enhanced translational regulation and ER stress sensing, however they were unable to trigger UPR-related transcriptional responses. This was accompanied by diminished ATF6 nuclear localization in stressed senescent cells. Finally, we revealed a deterioration of proteasome function in senescence following heat stress, which did not recover upon return to normal temperature. Together, our data unraveled a deterioration in the ability to mount dynamic stress transcriptional programs upon human senescence with broad implications on proteostasis control, and connected proteostasis decline to human aging. SignificanceProtein homeostasis (proteostasis), the balance between protein synthesis, folding, and degradation, is thought to deteriorate with age, and the prevalence of protein misfolding diseases, e.g. Alzheimers, Parkinsons etc., with human aging is increased. However, while in worms this phenomenon has been well established, in humans it remained unclear. Here we show that proteostasis is declined in human senescence, i.e. cellular aging. We found that while stress sensing is enhanced in senescent cells, and their response at the level of protein synthesis is intact, they fail to properly activate multiple programs required for stress adaptation at the level of gene transcription. Our findings support the notion that proteostasis decline may have major implications on human aging.

molecular biology↗

bioRxiv2018

Widespread PERK-dependent repression of ER targets in response to ER stress

The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated.\n\nUsing ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK -/- Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK -/- cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK -/- cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression gene expression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response.

molecular biology↗