Biology subjects

Roelofs, J.

Publications and source records attributed to Roelofs, J..

7 recordsLinked to original sources

bioRxiv2025

STI1 domain dynamically engages transient helices in disordered regions to drive self-association and phase separation of yeast ubiquilin Dsk2

Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation in vitro. Using NMR spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically-disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces the propensity for Dsk2 to phase separate. In vivo, removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes. HighlightsO_LIThe intrinsically disordered regions of Dsk2 harbor transient helices that regulate protein properties via interactions with the STI1 domain. C_LIO_LIThe STI1 domain is a significant driver of Dsk2 self-association and phase separation in vitro. C_LIO_LIDsk2 colocalizes with ubiquitinated substrates and proteasome in reconstituted condensates. C_LIO_LIAbsence of Dsk2 STI1 domain in stressed yeast cells promotes formation of proteasome condensates coupled with upregulation of polyubiquitinated substrates. C_LI

biochemistry↗

bioRxiv2025

NNMT promotes tubular senescence and fibrosis in chronic kidney disease

Chronic kidney disease (CKD) is a major global health issue, projected to become the fifth leading cause of mortality by 2040. Renal tubular cell senescence is a key driver of kidney fibrosis, the final manifestation of CKD. However, current treatment strategies, do not target senescent cells, as the underlying mechanisms driving this dysfunctional phenotype remain poorly described. Here, we identify nicotinamide-N-methyltransferase (NNMT), as a critical mediator of tubular senescence and fibrosis in CKD. Using human RNAseq profiles of CKD, we show that NNMT expression in the renal tubulointerstitium is strongly associated with CKD pathology and transcriptional signatures of cellular senescence. In human diabetic kidney disease biopsies, NNMT levels correlate with the senescence marker p21, kidney function decline, and fibrosis. Spatial transcriptomics further highlights that NNMT-positive tubules are senescent, fibrotic, and surrounded by a pro-inflammatory microenvironment. Preclinical models of early-stage CKD, show upregulation of NNMT and association with senescence. Overexpression of NNMT in TGF-{beta}-stimulated tubular epithelial cells promotes senescence and partial epithelial-to-mesenchymal transition (EMT), while inhibition of NNMT in kidney cells and organoids is protective. Altogether, we identify NNMT as a novel therapeutic target in the early stages of CKD with the potential to reduce tubular senescence, fibrosis and significantly slow disease progression.

cell biology↗

bioRxiv2024

Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle

Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.

biochemistry↗

bioRxiv2023

In vitro reconstitution of the M.tb proteasome core particle reveals conserved aspects of bacterial proteasome assembly

According to the WHO, one in three people in the world has a latent tuberculosis infection. Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (M.tb). The development of multi-drug resistant (MDR) tuberculosis indicates a need for novel treatments. Hence, it is important to find a second line of treatment for patients infected with MDR tuberculosis. The proteasome is known to be necessary for survival under stress and pathogenicity in M.tb. However, our ability to use the proteasome as drug target has been limited by our abilities to screen for inhibitor compounds in vitro. The proteasome is a protease complex that degrades proteins and is crucial for the maintenance of protein homeostasis within cells. Like many protein complexes, the proteasome must assemble into a specific quaternary structure in order to be active. Specifically, the proteolytically-active proteasome Core Particle (CP) consists of 28 subunits (14 and 14 {beta}) that must assemble into a barrel-like structure in order to become catalytically active. Hence, understanding the assembly process in not only important from a basic cell biological perspective, but may also serve as the basis for the discovery of novel assembly inhibitors. In this study, we have established for the first time a protocol to express and purify the M.tb and {beta} subunits separately in vitro. The subunits are soluble monomers on purification and only assemble into active CPs upon reconstitution. Our assembly experiments revealed that M.tb CP assembly pathway is almost certainly identical to that seen in previous experiments on the CP from the bacterium Rhodococcus erythropolis (R.e), but assembly in M.tb is much slower. Interestingly, we found that subunits from M.tb and R.e spontaneously self-assembled into active hybrid proteasomes on reconstitution with each other, despite having only 65% sequence similarity. Our work thus strongly suggests that the CP assembly pathway is conserved across bacteria, and the ability to perform in vitro assembly experiments on the M.tb proteasome opens up the possibility of performing critical experiments, including screening for potential molecules that could inhibit assembly, directly in this clinically-relevant organism.

biochemistry↗

bioRxiv2023

Proteasome condensate formation is driven by multivalent interactions with shuttle factors and K48-linked ubiquitin chains

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome (Rpn1, Rpn10, and Rpn13) are critical under different condensate inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes. SignificanceStress conditions can cause the relocalization of proteasomes to condensates in yeast as well as mammalian cells. Our work shows that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains, the proteasome binding shuttle factors Rad23 and Dsk2 and proteasome intrinsic ubiquitin receptors. Here, different receptors are critical for different condensate inducers. These results indicate distinct condensates can form with specific functionality. Our identification of key factors involved in the process is crucial for understanding the function of proteasome relocalization to condensates. We propose that cellular accumulation of substrates with long ubiquitin chains results in the formation of condensates comprising those ubiquitinated substrates, proteasomes, and proteasome shuttle factors, where the ubiquitin chains serve as the scaffold for condensate formation.

cell biology↗

bioRxiv2022

Proteasome granular localization is regulated through mitochondrial respiration and kinase signaling

In yeast, proteasomes are enriched in cell nuclei where they execute important cellular functions. Nutrient-stress can change this localization indicating proteasomes respond to the cells metabolic state. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules (PSGs). Surprisingly, we observed strongly reduced PSG levels when cells had active cellular respiration prior to starvation. This suggests the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function we observed proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a MAP kinase cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, while dispensable for granule formation following carbon starvation. We propose a model where mitochondrial activity promotes proteasome nuclear localization. SummaryWe determined a role for mitochondrial respiration in regulating proteasome granule formation and identified the cell integrity MAP kinase pathway and Snf1 kinase as regulatory factors.

cell biology↗

bioRxiv2021

Proteasome autophagy is specifically regulated and requires factors dispensible for general autophagy

Changing physiological conditions can increase the need for protein degradative capacity in eukaryotic cells. Both the ubiquitin-proteasome system and autophagy contribute to protein degradation. However, proteasomes are also an autophagy substrate. Thus, these processes must be differentially regulated depending on the physiological conditions presented. The signals and molecular mechanisms that govern proteasome autophagy are only partly elucidated. Our data indicate that chemical inhibition of TORC1 with rapamycin induces a bi-phasic response where proteasome levels are upregulated followed by an autophagy-dependent reduction. Surprisingly, several conditions that result in inhibited TORC1 exclusively induce proteasome autophagy (i.e. without any proteasome upregulation), suggesting a convergence of signals upstream of proteasome autophagy under different physiological conditions. Indeed, several conditions that activate general autophagy did not induce proteasome autophagy further distinguishing between proteasome autophagy and general autophagy. Consistent with this, we found that Atg11, the receptor for selective autophagy, and the map kinases Mpk1, Mkk1, and Mkk2, all play a role in autophagy of proteasomes, while they are dispensible for general autophagy. In all, our data provide new insights into the molecular regulation of proteasome autophagy by demonstrating that these complexes are specifically regulated under different autophagy inducing conditions.

biochemistry↗