Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

A genome-wide spectrum of tandem repeat expansions in 338,963 humans.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Structural evolution of fibril polymorphs during amyloid assembly.

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.

A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

A diterpene synthase from the sandfly Lutzomyia longipalpis produces the pheromone sobralene.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which ß-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-ß-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Elimination of virus-like particles reduces protein aggregation and extends replicative lifespan in Saccharomyces cerevisiae.

A major consequence of aging and stress, in yeast to humans, is an increased accumulation of protein aggregates at distinct sites within the cells. Using genetic screens, immunoelectron microscopy, and three-dimensional modeling in our efforts to elucidate the importance of aggregate annexation, we found that most aggregates in yeast accumulate near the surface of mitochondria. Further, we show that virus-like particles (VLPs), which are part of the retrotransposition cycle of Ty elements, are markedly enriched in these sites of protein aggregation. RNA interference-mediated silencing of Ty expression perturbed aggregate sequestration to mitochondria, reduced overall protein aggregation, mitigated toxicity of a Huntington's disease model, and expanded the replicative lifespan of yeast in a partially Hsp104-dependent manner. The results are in line with recent data demonstrating that VLPs might act as aging factors in mammals, including humans, and extend these findings by linking VLPs to a toxic accumulation of protein aggregates and raising the possibility that they might negatively influence neurological disease progression.

Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid.

α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.

An aggregated mitochondrial distribution in preimplantation embryos disrupts nuclear morphology, function, and developmental potential.

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.

The role of shear forces in primary and secondary nucleation of amyloid fibrils.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

Proteolytic stability and aggregation in a key metabolic enzyme of bacteria.

Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. ß-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Toward the quantification of α-synuclein aggregates with digital seed amplification assays.

The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.

Self-replication of Aß42 aggregates occurs on small and isolated fibril sites.

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer's A[Formula: see text] amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A[Formula: see text] fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

Helical sulfonyl-γ-AApeptides modulating Aß oligomerization and cytotoxicity by recognizing Aß helix.

In contrast to prevalent strategies which make use of ß-sheet mimetics to block Aß fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aß13-26 and stabilized Aß conformation to avoid forming the neurotoxic Aß oligomeric ß-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aß oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aß42 and induce α-helix conformation in Aß. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aß aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aß-mediated cytotoxicity even in the presence of pre-formed Aß aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aß42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aß42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aß into the off-pathway helical secondary structure, thereby preventing the formation of Aß oligomerization, fibrillation and rescuing Aß induced cell cytotoxicity.

Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

Computational insights into the cross-talk between medin and Aß: implications for age-related vascular risk factors in Alzheimer's disease.

The aggregation of medin forming aortic medial amyloid is linked to arterial wall degeneration and cerebrovascular dysfunction. Elevated levels of arteriolar medin are correlated with an increased presence of vascular amyloid-ß (Aß) aggregates, a hallmark of Alzheimer's disease (AD) and vascular dementia. The cross-interaction between medin and Aß results in the formation of heterologous fibrils through co-aggregation and cross-seeding processes both in vitro and in vivo. However, a comprehensive molecular understanding of the cross-interaction between medin and Aß-two intrinsically disordered proteins-is critically lacking. Here, we employed atomistic discrete molecular dynamics simulations to systematically investigate the self-association, co-aggregation and also the phenomenon of cross-seeding between these two proteins. Our results demonstrated that both Aß and medin were aggregation prone and their mixture tended to form ß-sheet-rich hetero-aggregates. The formation of Aß-medin hetero-aggregates did not hinder Aß and medin from recruiting additional Aß and medin peptides to grow into larger ß-sheet-rich aggregates. The ß-barrel oligomer intermediates observed in the self-aggregations of Aß and medin were also present during their co-aggregation. In cross-seeding simulations, preformed Aß fibrils could recruit isolated medin monomers to form elongated ß-sheets. Overall, our comprehensive simulations suggested that the cross-interaction between Aß and medin may contribute to their pathological aggregation, given the inherent amyloidogenic tendencies of both medin and Aß. Targeting medin, therefore, could offer a novel therapeutic approach to preserving brain function during aging and AD by improving vascular health.

Effective multi-modal clustering method via skip aggregation network for parallel scRNA-seq and scATAC-seq data.

In recent years, there has been a growing trend in the realm of parallel clustering analysis for single-cell RNA-seq (scRNA) and single-cell Assay of Transposase Accessible Chromatin (scATAC) data. However, prevailing methods often treat these two data modalities as equals, neglecting the fact that the scRNA mode holds significantly richer information compared to the scATAC. This disregard hinders the model benefits from the insights derived from multiple modalities, compromising the overall clustering performance. To this end, we propose an effective multi-modal clustering model scEMC for parallel scRNA and Assay of Transposase Accessible Chromatin data. Concretely, we have devised a skip aggregation network to simultaneously learn global structural information among cells and integrate data from diverse modalities. To safeguard the quality of integrated cell representation against the influence stemming from sparse scATAC data, we connect the scRNA data with the aggregated representation via skip connection. Moreover, to effectively fit the real distribution of cells, we introduced a Zero Inflated Negative Binomial-based denoising autoencoder that accommodates corrupted data containing synthetic noise, concurrently integrating a joint optimization module that employs multiple losses. Extensive experiments serve to underscore the effectiveness of our model. This work contributes significantly to the ongoing exploration of cell subpopulations and tumor microenvironments, and the code of our work will be public at https://github.com/DayuHuu/scEMC.