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It is now well established that the movement of the eyes, which occurs constantly even during fixation, tends to “freeze” in response to perceptual events, with a magnitude and time course that depends on the stimulus properties, attention, and anticipation. This “freeze” or oculomotor inhibition (OMI) was found for microsaccades, blinks, smooth-pursuit, and catch-up saccades; yet remains unclear whether it also applies to ocular drift. Since video-based eye-trackers are known to produce positional artifacts, we used here data from a high-speed and precision retinal imaging eye-tracker (FreezEye Tracker, FET). The observers (n = 15) watched a series of flashed Gabor patches, with different spatial frequency and contrast while their eyes were tracked. We analyzed the data by removing the saccades, aligning the traces, and computing four drift measures relative to the stimulus onset: (1) velocity, (2) area, (3) diffusion, and (4) heat map. We found that all measures produced a highly significant modulation over time. The overall drift velocity, area, and diffusion followed the microsaccade inhibition pattern, whereas the heat map peaks showed the opposite pattern. The drift inhibition was also stimulus dependent, showing shorter onset and prolonged release estimates for more salient stimuli, matching the microsaccade OMI for the onset but showing the opposite for the release. Overall, the results confirm that the oculomotor inhibition effect can be generalized for ocular drift, but its opposite stimulus dependency for inhibition release and the shifted time course may suggest a complex interdependency between drift and saccades.

SUMMARYProtein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms mislocalized cytoplasmic aggregates in the brains of old killifish and mice. Interestingly, DDX5’s prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the protein’s ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.

SUMMARYProtein aggregation is a hallmark of age-related neurodegeneration. Yet, aggregation during normal aging and in tissues other than the brain is poorly understood. Here we leverage the African turquoise killifish to systematically profile protein aggregates in seven tissues of an aging vertebrate. Age-dependent aggregation is strikingly tissue-specific, and not simply driven by protein expression differences. Experimental interrogation, combined with machine learning, indicates that this specificity is linked to both protein-autonomous biophysical features and tissue-selective alterations in protein quality control. Co-aggregation of protein quality control machinery during aging may further reduce proteostasis capacity, exacerbating aggregate burden. A segmental progeria model with accelerated aging in specific tissues exhibits selectively increased aggregation in these same tissues. Intriguingly, many age-related protein aggregates arise in wild-type proteins that, when mutated, drive human diseases. Our data chart a comprehensive landscape of protein aggregation during aging and reveal strong, tissue-specific associations with dysfunction and disease.HIGHLIGHTSTissue-specific protein aggregation is prevalent during vertebrate agingBoth protein biophysical properties and tissue-specific protein homeostasis patterns impact aggregationA segmental progeria model with accelerated aging exhibits selectively increased protein aggregation in affected tissuesMany aggregates that accumulate during physiological aging are linked to disease

Our eyes are never still, but tend to "freeze" in response to stimulus onset. This effect is termed "oculomotor inhibition" (OMI); its magnitude and time course depend on the stimulus parameters, attention, and expectation. We previously showed that the time course and duration of microsaccade and spontaneous eye-blink inhibition provide an involuntary measure of low-level visual properties such as contrast sensitivity during fixation. We investigated whether this stimulus-dependent inhibition also occurs during smooth pursuit, for both the catch-up saccades and the pursuit itself. Observers followed a target with continuous back-and-forth horizontal motion while a Gabor patch was briefly flashed centrally with varied spatial frequency and contrast. Catch-up saccades of the size of microsaccades had a similar pattern of inhibition as microsaccades during fixation, with stronger inhibition onset and faster inhibition release for more salient stimuli. Moreover, a similar stimulus dependency of inhibition was shown for pursuit latencies and peak velocity. Additionally, microsaccade latencies at inhibition release, peak pursuit velocities, and latencies at minimum pursuit velocity were correlated with contrast sensitivity. We demonstrated the generality of OMI to smooth pursuit for both microsaccades and the pursuit itself and its close relation to the low-level processes that define saliency, such as contrast sensitivity.

Protein aggregation, particularly in its prion-like form, has long been thought to be detrimental. However, recent studies have identified multiple instances where protein aggregation is important for normal physiological functions. Combining mass spectrometry and cell biological approaches, we developed a strategy for the identification of protein aggregates in cell lysates. We used this approach to characterize prion-based traits in pathogenic strains of the yeast Saccharomyces cerevisiae isolated from immunocompromised human patients. The proteins that we found, including the metabolic enzyme Cdc19, the translation elongation factor Yef3 and the fibrillarin homologue Nop1, are known to assemble under certain physiological conditions. Yet, such assemblies have not been reported to be stable or heritable. Our data suggest that some proteins which aggregate in response to stress have the capacity to acquire diverse assembled states, certain ones of which can be propagated across generations in a form of protein-based epigenetics. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

Ubiquitin-binding domains (UBDs) differentially recognize ubiquitin (ub) modifications. Some of them specifically bind mono-ub, as has been shown for the CUE domain. Interestingly, so far no significant ubiquitin binding has been observed for the CUE domain of yeast Cue1p. Cue1p is receptor and activator of the ubiquitin-conjugating enzyme Ubc7p. It integrates Ubc7p into endoplasmic reticulum (ER) membrane-bound ubiquitin ligase complexes, and thus, it is crucial for ER-associated protein degradation (ERAD). Here we show that the CUE domain of Cue1p binds ubiquitin chains, which is pivotal for the efficient formation of K48-linked polyubiquitin chains in vitro. Mutations that abolish ubiquitin binding by Cue1p affect the turnover of ERAD substrates in vivo. Our data strongly imply that the CUE domain facilitates substrate ubiquitylation by stabilizing growing ubiquitin chains at the ERAD ubiquitin ligases. Hence, we demonstrate an unexpected function of a UBD in the regulation of ubiquitin chain synthesis.

As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone, and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin-chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly-tagged cargo. We propose a mechanism for a malleable ubiquitin-signal that depends both on chain-length and combination of receptors to produce tetra-ubiquitin as an efficient signal threshold.

Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine 48-linked ubiquitin is essential for marking targets for proteasomal degradation, but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine 48 and lysine 63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine 48 linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.

Negative feedback is among the key mechanisms for regulating receptor tyrosine kinase (RTK) signaling. Human Sef, a recently identified inhibitor of RTK signaling, encodes different isoforms, including a membrane spanning (hSef-a) and a cytosolic (hSef-b) isoform. Previously, we reported that hSef-b inhibited fibroblast proliferation and prevented the activation of mitogen-activated protein kinase (MAPK), without affecting protein kinase B/Akt or p38 MAPK. Conflicting results were reported concerning hSef-a inhibition of MAPK activation, and the effect of hSef-a on other RTK-induced signaling pathways is unknown. Here we show that, in fibroblasts, similar to hSef-b, ectopic expression of hSef-a inhibited fibroblast growth factor-induced cell proliferation. Unlike hSef-b, however, the growth arrest was mediated via a MAPK-independent mechanism, and was accompanied by elevated p38 MAPK phosphorylation and inhibition of protein kinase B/Akt. In addition, hSef-a, but not hSef-b, mediated apoptosis in fibroblast growth factor-stimulated cells. Chemical inhibitor of p38 MAPK abrogated the effect of hSef-a on apoptosis. In epithelial cells, ectopic expression of hSef-a inhibited the activation of MAPK, whereas down-regulation of endogenous hSef-a significantly increased MAPK activation and accelerated growth factor-dependent cell proliferation. These results indicate that hSef-a is a multifunctional negative modulator of RTK signaling and clearly demonstrate that hSef-a can inhibit the activation of MAPK, although in a cell type-specific manner. Moreover, the differences between the activities of hSef-a and hSef-b suggest that hSef isoforms can control signal specificity and subsequent cell fate by utilizing different mechanisms to modulate RTK signaling.

EMBO J 28, 1867–1877 (2009); published online 08 July 2009 [PMC free article] [PubMed] Polyubiquitin (polyUb) is a diverse signal in terms of both chain length and linkage type (Ikeda and Dikic, 2008). Chains can be formed through covalent conjugation of ubiquitin (Ub) to any of the seven lysine residues on the preceding Ub (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, or Lys63), and in some instances two Ub molecules can simultaneously modify two lysine residues on a single Ub resulting in branched chains (Kim et al, 2007). Ubiquitin chain-length and linkage type, along with affinity for proteasome receptors and ease of deubiquitination, all contribute towards setting substrate hierarchy. Hence, polyUb-binding proteins influence the specificity and efficiency of intra-cellular proteolysis. One such auxiliary factor, S5a (or Rpn10), partakes in shuttling polyUb conjugates, limiting the access of competing substrate carriers, and in anchoring Ub chains at the proteasome (Deveraux et al, 1994; Glickman et al, 1998; Matiuhin et al, 2008). It now seems that S5a/Rpn10 also functions in upstream events by blocking the synthesis of low-priority forked chains and promoting the formation of unbranched chains with high affinity for the proteasome (Kim et al, 2009). In an attempt to understand why some conjugation reactions generate polyubiquitinated substrates that are poorly degraded, and how Ub-binding proteins support proteosomal degradation, Kim et al have added purified S5a to a coupled ubiquitination–degradation assay. The presence of S5a in the reaction containing a substrate (luciferase), ubiquitination enzymes (an E2, UbcH5, and the U-box E3 ligase CHIP) and 26S proteasome significantly increased the rate of luciferase degradation. S5a had a similar effect on the degradation of another substrate (troponin I) ubiquitinated by UbcH5 and another E3 ubiquitin ligase (MuRF1). The yeast homologue of S5a, Rpn10, enhanced the proteasomal degradation of troponin I to the same extent as S5a. In order to understand the source of proteolysis enhancement, S5a was then added to the reaction mix after the formation of ubiquitin conjugates. This move actually inhibited the degradation of the luciferase substrate, indicating that S5a is involved in early steps of targeting proteins for degradation. The use of mutated ubiquitin restricted in the linkages it can form (K48R, K11R, K29R, or K63R) did not alter the effect of S5a on degradation, indicating that degradation is not achieved by increasing the levels of any specific chain linkage. Nevertheless, mass spectrometry analysis of conjugates formed by CHIP with UbcH5 showed a decrease in forked linkages when S5a was added to the ubiquitination reaction. Forked chains are those in which two Ub molecules are linked to two adjacent lysines on the preceding Ub molecule (in essence, rather than forming elongated chains, some polyUbs might exist as a branched bush; see Figure 1A). It is important to note that because of technical limitations only simultaneous modification on adjacent lysines (K6/11, K27/29, or 29/33) was assayed in the accompanying study. The effect of S5a on the formation of forked chains was also detected during ubiquitination by the MuRF1 and UbcH5 pair of enzymes. Hydrolysis of substrates linked to homogenous non-forked Ub chains was not enhanced by addition of S5a and the authors therefore conclude that S5a interacts with the growing polyUb chain to prevent fork formation. Figure 1 The ubiquitin-binding protein, S5a/Rpn10, trails ubiquitin conjugates along their trajectory. (A) A general scheme of the ubiquitin–proteasome pathway updated with new results described by Kim et al (2009) in this issue. Ubiquitination enzymes ... What hurdle do forked Ub chains pose on protein degradation? Part of the effect of S5a seems to stem from the difficulty in processing (or deubiquitinating) forked chains. In an elegant set of experiments, the authors go on to show that forked Ub chains bind poorly to 26S, in contrast to non-forked ones. In a competition assay, luciferase linked to mixed forked Ub chains (ubiquitinated in the absence of S5a) was unable to inhibit the proteasomal degradation of troponin I linked to Lys63 or Lys48 chains, whereas luciferase linked to Lys63 chains or mixed non-forked chains (ubiquitinated in the presence of S5a) caused a significant decrease in the degradation of troponin I bound to homogeneous chains. Furthermore, in a binding assay, MuRF1 autoubiquitinated in the presence of S5a showed higher affinity for purified 26S than MuRF1 ubiquitinated in the absence of S5a. Poor anchoring to the proteasome (by S5a or another receptor; Figure 1A) might be the cause for the apparent stability and slow rate of processing or proteolysis of these conjugates. The accompanying manuscript by Kim et al (2009) opens up a porthole to an exciting new layer of complexity in directing the ubiquitination process. Ubiquitin chain length and linkage type, along with the affinity for proteasome receptors and ease of deubiquitination, all contribute towards setting substrate hierarchy (Figure 1A). To these selection processes, one can now add a new checkpoint: at early stages of ubiquitination S5a limits the formation of forked chains, in which chains are extended at more than one lysine on a given Ub. Yet, as with many new observations, some amount of caution should be exercised when considering the general implications. So far branched chains have been identified mostly in vitro and their formation is strongly depended on the E2 used in the ubiquitination reaction. Among all possible branched modifications, only three forks at adjacent lysines on a single ubiquitin (K6/11, K27/29, and K29/33) have been documented. An accurate quantification of branched ubiquitin relative to total Ub-in-chains is yet to be carried out, although the assumption is that the ratio of forked modifications over extended chains is low (Kim et al, 2009). Furthermore, S5a UIM binds Ub through a patch of hydrophobic residues (Walters et al, 2002), leaving most lysine residues exposed for unrestricted conjugation to subsequent Ub (Figure 1B). This implies an intricate mechanism for S5a to block synthesis of forked chains, involving interactions between multiple components of the synthesis machinery, perhaps by getting polyubiquitinated itself, thus deflecting the imprecise ubiquitination of the substrate (Kim et al, 2009). Nonconformity in ubiquitin polymerization—e.g. arborization in contrast to extension—might lead to accumulation of highly stable ubiquitin conjugates that are poorly recognized and slow to be removed. As previously shown in vivo, expression of Rpn10 or its UIM domain alone was able to reverse the accumulation of poorly turned-over polyUb conjugates under an induced stress condition (Matiuhin et al, 2008). The corrective properties of S5a in directing proper chain formation and prioritizing proteasome-bound substrates might be useful for enhancing protein turnover or overcoming stress conditions associated with malfunctions in the ubiquitin–proteasome system.

International audience; Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.

Receptor tyrosine kinases (RTKs) control a multitude of biological processes and are therefore subjected to multiple levels of regulation. Negative feedback is one of the mechanisms that provide an effective means to control RTK-mediated signaling. Sef has recently been identified as a specific antagonist of fibroblast growth factor (FGF) signaling in zebrafish and subsequently in mouse and human. Sef encodes a putative type I transmembrane protein that antagonizes the Ras/mitogen-activated protein kinase pathway in all three species. Mouse Sef was also shown to inhibit the phosphatidylinositol 3-kinase pathway. We show here that an alternative splicing mechanism generates an isoform of human Sef, hSef-b, which unlike the previously reported Sef (hSef-a) is a cytosolic protein. Contrary to hSef-a, which is ubiquitously expressed, hSef-b transcripts display a restricted pattern of expression in human tissues. hSef-b inhibits FGF-induced cell proliferation and prevents the activation of mitogen-activated protein kinase without affecting the upstream component MAPK kinase. Furthermore, hSef-b does not antagonize FGF induction of the phosphatidylinositol 3-kinase pathway. In addition to the effects on FGF signaling, hSef-b inhibited cellular response to platelet-derived growth factor but not other RTK ligands. Therefore, alternative splicing of the hSef gene expands the Sef feedback inhibition repertoire of RTK signaling.