Calmodulin regulates protease versus co-chaperone activity of a metacaspase.

Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

SMER28 binding to VCP/p97 enhances both autophagic and proteasomal neurotoxic protein clearance.

The ability to maintain a functional proteome by clearing damaged or misfolded proteins is critical for cell survival, and aggregate-prone proteins accumulate in many neurodegenerative diseases, such as Huntington, Alzheimer, and Parkinson diseases. The removal of such proteins is mainly mediated by the ubiquitin-proteasome system and autophagy, and the activity of these systems declines in disease or with age. We recently found that targeting VCP/p97 with compounds like SMER28 enhances macroautophagy/autophagy flux mediated by the increased activity of the PtdIns3K complex I. Additionally, we found that SMER28 binding to VCP stimulates aggregate-prone protein clearance via the ubiquitin-proteasome system. This concurrent action of SMER28 on both degradation pathways resulted in the selective decrease in disease-causing proteins but not their wild-type counterparts. These results reveal a promising mode of VCP activation to counteract the toxicity caused by aggregate-prone proteins.

Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance.

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.

Large organellar changes occur during mild heat shock in yeast.

When the temperature is increased, the heat-shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilised for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes and morphologies in over 2100 electron micrographs, a major restructuring of the internal architecture of the cell during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of the morphology of some organelles (such as the vacuole) were observed. Multivesicular bodies grew by almost 70%, indicating a previously unknown involvement in the heat-shock response. A previously undescribed electron-translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.

VCP/p97 modulates PtdIns3P production and autophagy initiation.

VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.

VCP/p97 regulates Beclin-1-dependent autophagy initiation.

Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.

The role of sodium chloride in the sensory and physico-chemical properties of sweet biscuits.

Salt is included in many foods which consumers do not regard as salty. This "hidden-salt" may offer functional benefits but is often overlooked in sodium reduction strategies. This study investigated its role in shortbread-like sweet biscuits (1.05 g NaCl/100 g). Sensory tests revealed significant flavour and texture differences after a salt reduction of 33% (0.86 g/ 100 g). This was explained by differences in the partitioning of hydrophobic aroma compounds into the headspace and a significant impact on structure. Texture analysis and X-ray-µCT measurements revealed a reduced hardness with larger and more air cells in salt-reduced biscuits. It is suggested that salt impacts on cereal proteins by altering their aggregation around flour particles and at bubble walls and that slower water loss occurs in salted matrices during baking. Hence, this study revealed the key properties significantly affected by salt reduction and proposes an explanation which will help to develop a targeted "hidden-salt" reduction strategy.

mTORC2 Assembly Is Regulated by USP9X-Mediated Deubiquitination of RICTOR.

The mechanistic target of rapamycin complex 2 (mTORC2) controls cell metabolism and survival in response to environmental inputs. Dysregulation of mTORC2 signaling has been linked to diverse human diseases, including cancer and metabolic disorders, highlighting the importance of a tightly controlled mTORC2. While mTORC2 assembly is a critical determinant of its activity, the factors regulating this event are not well understood, and it is unclear whether this process is regulated by growth factors. Here, we present data, from human cell lines and mice, describing a mechanism by which growth factors regulate ubiquitin-specific protease 9X (USP9X) deubiquitinase to stimulate mTORC2 assembly and activity. USP9X removes Lys63-linked ubiquitin from RICTOR to promote its interaction with mTOR, thereby facilitating mTORC2 signaling. As mTORC2 is central for cellular homeostasis, understanding the mechanisms regulating mTORC2 activation toward its downstream targets is vital for our understanding of physiological processes and for developing new therapeutic strategies in pathology.

Erratum: Author Correction: Mendelian neurodegenerative disease genes involved in autophagy.

[This corrects the article DOI: 10.1038/s41421-020-0158-y.].

Mendelian neurodegenerative disease genes involved in autophagy.

The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases. Here, we review mutations that have been identified in genes involved in autophagy and their associations with neurodegenerative diseases.

Incorporation of a novel leguminous ingredient into savoury biscuits reduces their starch digestibility: Implications for lowering the Glycaemic Index of cereal products.

Many carbohydrate foods contain starch that is rapidly digested and elicits a high Glycaemic Index. A legume ingredient (PulseON®) rich in Type 1 resistant starch (RS1) was recently developed; however, its potential as a functional ingredient when processed into a food product required assessment. PulseON® was used to replace 0, 25, 50, 75, and 100% of the wheat flour in a savoury biscuit recipe. In vitro starch digestion kinetics of biscuits and water-holding properties of ingredients were assessed. The RS1 in PulseON® did not appear to be structurally compromised during biscuit making. Replacing 50% wheat flour with PulseON® reduced the starch hydrolysis index of biscuits by nearly 60%. This seems to result from the ingredients' impact on water availability for starch gelatinisation. Overall, these findings highlight the potential of using biscuits as a food vehicle for PulseON® to increase consumer intakes of legume protein, dietary fibre, and potentially low glycaemic starch.

Non-chemically modified waxy rice starch stabilised wow emulsions for salt reduction.

Water-in-oil-in-water emulsions containing an internalised salt solution were stabilised with non-chemically modified waxy rice starch (WRS), and octinyl succinic anhydride (OSA) as reference, to release salt during oral processing due to amylase-induced destabilisation. Salt levels were 1.5 g salt and 0.47 g salt per 100 g external and internal aqueous phases, respectively. Variables were the starch content (2, 3, 4 g per 100 g emulsion; 20 g oil per 100 g emulsion), level of polyglycerol polyricinoleate (PGPR) as a lipophilic emulsifier (0.29, 0.57 g per 100 g emulsion) and ambient-pressure processing temperature for WRS gelatinisation, the non-chemical modification process, (75 ± 3, 88 ± 5 °C). OSA starch was used under previously applied conditions (2, 3, 4 g starch, 0.57 g PGPR per 100 g emulsion, 25 ± 5 °C). Emulsions were stable for three months, except OSA and lower level PGPR low temperature processed WRS emulsions lost salt into the external emulsion phase. One day after processing, encapsulation efficiency (EE) was as predicted from the composition for OSA emulsions, while at the same PGPR content an external aqueous phase was incorporated into the oil droplets of the WRS emulsion increasing EE. Salt release was assessed in vitro and through sensory evaluation using paired comparison testing. The results revealed that the efficacy of this salt reduction approach was enhanced for gelatinised WRS compared to OSA starch stabilised emulsions. Consumer tests on a tomato soup, to validate this salt reduction approach for a real food, revealed a possible 25% salt reduction, compared to current UK products.

Correction to: Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.

Reference 45 was incorrect in the original version of this article.

Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.

Autophagy is a conserved intracellular degradation pathway essential for protein homeostasis, survival and development. Defects in autophagic pathways have been connected to a variety of human diseases, including cancer and neurodegeneration. In the process of macroautophagy, cytoplasmic cargo is enclosed in a double-membrane structure and fused to the lysosome to allow for digestion and recycling of material. Autophagosome formation is primed by the ULK complex, which enables the downstream production of PI(3)P, a key lipid signalling molecule, on the phagophore membrane. The PI(3)P is generated by the PI3 kinase (PI3K) complex, consisting of the core components VPS34, VPS15 and Beclin 1. Beclin 1 is a central player in autophagy and constitutes a molecular platform for the regulation of autophagosome formation and maturation. Post-translational modifications of Beclin 1 affect its stability, interactions and ability to regulate PI3K activity, providing the cell with a plethora of strategies to fine-tune the levels of autophagy. Being such an important regulator, Beclin 1 is a potential target for therapeutic intervention and interfering with the post-translational regulation of Beclin 1 could be one way of manipulating the levels of autophagy. In this review, we provide an overview of the known post-translational modifications of Beclin 1 that govern its role in autophagy and how these modifications are maintained by input from several upstream signalling pathways. ▓.

Reduction of enthalpy relaxation in gelatine films by addition of polyols.

The aim of this study was to evaluate the effect of plasticisers with different molecular weights (glycerol and sorbitol) on the structural relaxation kinetics of bovine gelatine films stored under the glass transition temperature (Tg). Plasticisers were tested at weight fractions of 0.0, 0.06 and 0.10. Films conditioned in environments under ∼44% relative humidity gave moisture contents (w/w) in the range 0.14-0.18. The enthalpy relaxation (ΔH) was determined using differential scanning calorimetry (DSC). Samples used had Tg values in the range 24-49 °C. After removing the thermal history (30 °C above Tg, 15 min), samples were isothermally stored at 10 °C below Tg for between 2 and 80 h. The addition of plasticisers induced a significant reduction in the rate of structural relaxation. The linearisation of ΔH by plotting against the logarithm of ageing time showed a reduction in the slope of samples plasticised with both polyols. The reduction in relaxation kinetics may be related to the ability of polyols to act as enhancers of molecular packing, as recently reported using positron spectroscopy (PALS). However, a direct correlation between the relaxation kinetics and the plasticiser's molecular weight could not be established, suggesting that this phenomenon may be governed by complex molecular gelatin-plasticiser-water interactions.

Restricted access: spatial sequestration of damaged proteins during stress and aging.

The accumulation of damaged and aggregated proteins is a hallmark of aging and increased proteotoxic stress. To limit the toxicity of damaged and aggregated proteins and to ensure that the damage is not inherited by succeeding cell generations, a system of spatial quality control operates to sequester damaged/aggregated proteins into inclusions at specific protective sites. Such spatial sequestration and asymmetric segregation of damaged proteins have emerged as key processes required for cellular rejuvenation. In this review, we summarize findings on the nature of the different quality control sites identified in yeast, on genetic determinants required for spatial quality control, and on how aggregates are recognized depending on the stress generating them. We also briefly compare the yeast system to spatial quality control in other organisms. The data accumulated demonstrate that spatial quality control involves factors beyond the canonical quality control factors, such as chaperones and proteases, and opens up new venues in approaching how proteotoxicity might be mitigated, or delayed, upon aging.

Asymmetric Inheritance of Aggregated Proteins and Age Reset in Yeast Are Regulated by Vac17-Dependent Vacuolar Functions.

Age can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one. This phenomenon requires asymmetry-generating genes (AGGs) that govern the asymmetrical inheritance of aggregated proteins. Using a genome-wide imaging screen to identify AGGs in Saccharomyces cerevisiae, we discovered a previously unknown role for endocytosis, vacuole fusion, and the myosin-dependent adaptor protein Vac17 in asymmetrical inheritance of misfolded proteins. Overproduction of Vac17 increases deposition of aggregates into cytoprotective vacuole-associated sites, counteracts age-related breakdown of endocytosis and vacuole integrity, and extends replicative lifespan. The link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles. We also show that the protein disaggregase Hsp104 interacts physically with endocytic vesicle-associated proteins, such as the dynamin-like protein, Vps1, which was also shown to be required for Vac17-dependent sequestration of protein aggregates. These data demonstrate that two physiognomies of aging-reduced endocytosis and protein aggregation-are interconnected and regulated by Vac17.

Selective protein degradation ensures cellular longevity.

A previously unknown pathway can selectively degrade mitochondrial proteins in aged and stressed cells without destroying the organelle itself.

Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.