Gili Ben-Nissan

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by the core 20S proteasome itself. Degradation by the 20S proteasome does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the inherent structural disorder of the protein being degraded. Thus, proteins that contain unstructured regions due to oxidation, mutation, or aging, as well as naturally, intrinsically unfolded proteins, are susceptible to 20S degradation. Unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome, relatively little is known about the means by which 20S-mediated proteolysis is controlled. Here, we describe our current understanding of the regulatory mechanisms that coordinate 20S proteasome-mediated degradation, and highlight the gaps in knowledge that remain to be bridged.

A Mutually Inhibitory Feedback Loop between the 20S Proteasome and Its Regulator, NQO1

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.

The Parkinson’s-associated protein DJ-1 regulates the 20S proteasome

The Parkinsons-associated protein, DJ-1, is a highly conserved homodimer, ubiquitously expressed in cells. Here we demonstrate that DJ-1 is a 20S proteasome regulator. We show that DJ-1 physically binds the 20S proteasome and inhibits its activity, rescuing partially unfolded proteins from degradation. Consequently, DJ-1 stabilizes the cellular levels of 20S proteasome substrates, as we show for α-synuclein and p53. Furthermore, we demonstrate that following oxidative stress, DJ-1 is involved in the Nrf2-dependent oxidative stress response that leads to the upregulation of both the 20S proteasome and its regulator, NQO1. Overall, our results suggest a regulatory circuit in which DJ-1, under conditions of oxidative stress, both upregulates and inhibits the 20S proteasome, providing a rigorous control mechanism at a time when the 20S proteasome becomes the major proteolytic machinery. Such a tight regulation of the 20S proteasome may sustain the balance between the need to rapidly eliminate oxidatively damaged proteins and maintain the abundance of native, intrinsically unstructured proteins, which coordinate regulatory and signalling events.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

Exposing the subunit diversity within protein complexes: A mass spectrometry approach

Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes.

Dynamic Regulation of the COP9 Signalosome in Response to DNA Damage

ABSTRACT The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that participates in the regulation of the ubiquitin/26S proteasome pathway by controlling the function of cullin-RING-ubiquitin ligases. Impressive progress has been made in deciphering its critical role in diverse cellular and developmental processes. However, little is known about the underlying regulatory principles that coordinate its function. Through biochemical and fluorescence microscopy analyses, we determined that the complex is localized in the cytoplasm, nucleoplasm, and chromatin-bound fractions, each differing in the composition of posttranslationally modified subunits, depending on its location within the cell. During the cell cycle, the segregation between subcellular localizations remains steady. However, upon UV damage, a dose-dependent temporal shuttling of the CSN complex into the nucleus was seen, accompanied by upregulation of specific phosphorylations within CSN1, CSN3, and CSN8. Taken together, our results suggest that the specific spatiotemporal composition of the CSN is highly controlled, enabling the complex to rapidly adapt and respond to DNA damage.

CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

The highly conserved COP9 signalosome (CSN) complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein). We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex

Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.

Structural Characterization of Missense Mutations Using High Resolution Mass Spectrometry: A Case Study of the Parkinson’s-Related Protein, DJ-1

AbstractMissense mutations that lead to the expression of mutant proteins carrying single amino acid substitutions are the cause of numerous diseases. Unlike gene lesions, insertions, deletions, nonsense mutations, or modified RNA splicing, which affect the length of a polypeptide, or determine whether a polypeptide is translated at all, missense mutations exert more subtle effects on protein structure, which are often difficult to evaluate. Here, we took advantage of the spectral resolution afforded by the EMR Orbitrap platform, to generate a mass spectrometry-based approach relying on simultaneous measurements of the wild-type protein and the missense variants. This approach not only considerably shortens the analysis time due to the concurrent acquisition but, more importantly, enables direct comparisons between the wild-type protein and the variants, allowing identification of even subtle structural changes. We demonstrate our approach using the Parkinson’s-associated protein, DJ-1. Together with the wild-type protein, we examined two missense mutants, DJ-1A104T and DJ-1D149A, which lead to early-onset familial Parkinson’s disease. Gas-phase, thermal, and chemical stability assays indicate clear alterations in the conformational stability of the two mutants: the structural stability of DJ-1D149A is reduced, whereas that of DJ-1A104T is enhanced. Overall, we anticipate that the methodology presented here will be applicable to numerous other missense mutants, promoting the structural investigations of multiple variants of the same protein. Graphical Abstractᅟ