Nuclear Hsp104 safeguards the dormant translation machinery during quiescence.

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

Early fate decision for mitochondrially encoded proteins by a molecular triage.

Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded.

Newly imported proteins in mitochondria are particularly sensitive to aggregation.

AIM: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation. METHODS: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates. RESULTS: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation. CONCLUSION: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.

Reversible protein assemblies in the proteostasis network in health and disease.

While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed via liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.

Nuclear envelope budding and its cellular functions.

The nuclear pore complex (NPC) has long been assumed to be the sole route across the nuclear envelope, and under normal homeostatic conditions it is indeed the main mechanism of nucleo-cytoplasmic transport. However, it has also been known that e.g. herpesviruses cross the nuclear envelope utilizing a pathway entitled nuclear egress or envelopment/de-envelopment. Despite this, a thread of observations suggests that mechanisms similar to viral egress may be transiently used also in healthy cells. It has since been proposed that mechanisms like nuclear envelope budding (NEB) can facilitate the transport of RNA granules, aggregated proteins, inner nuclear membrane proteins, and mis-assembled NPCs. Herein, we will summarize the known roles of NEB as a physiological and intrinsic cellular feature and highlight the many unanswered questions surrounding these intriguing nuclear events.

Manganese-driven CoQ deficiency.

Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.

Sterol Metabolism Differentially Contributes to Maintenance and Exit of Quiescence.

Nutrient starvation initiates cell cycle exit and entry into quiescence, a reversible, non-proliferative state characterized by stress tolerance, longevity and large-scale remodeling of subcellular structures. Depending on the nature of the depleted nutrient, yeast cells are assumed to enter heterogeneous quiescent states with unique but mostly unexplored characteristics. Here, we show that storage and consumption of neutral lipids in lipid droplets (LDs) differentially impacts the regulation of quiescence driven by glucose or phosphate starvation. Upon prolonged glucose exhaustion, LDs were degraded in the vacuole via Atg1-dependent lipophagy. In contrast, yeast cells entering quiescence due to phosphate exhaustion massively over-accumulated LDs that clustered at the vacuolar surface but were not engulfed via lipophagy. Excessive LD biogenesis required contact formation between the endoplasmic reticulum and the vacuole at nucleus-vacuole junctions and was accompanied by a shift of the cellular lipid profile from membrane towards storage lipids, driven by a transcriptional upregulation of enzymes generating neutral lipids, in particular sterol esters. Importantly, sterol ester biogenesis was critical for long-term survival of phosphate-exhausted cells and supported rapid quiescence exit upon nutrient replenishment, but was dispensable for survival and regrowth of glucose-exhausted cells. Instead, these cells relied on de novo synthesis of sterols and fatty acids for quiescence exit and regrowth. Phosphate-exhausted cells efficiently mobilized storage lipids to support several rounds of cell division even in presence of inhibitors of fatty acid and sterol biosynthesis. In sum, our results show that neutral lipid biosynthesis and mobilization to support quiescence maintenance and exit is tailored to the respective nutrient scarcity.

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

Remodelling of Nucleus-Vacuole Junctions During Metabolic and Proteostatic Stress.

Cellular adaptation to stress and metabolic cues requires a coordinated response of different intracellular compartments, separated by semipermeable membranes. One way to facilitate interorganellar communication is via membrane contact sites, physical bridges between opposing organellar membranes formed by an array of tethering machineries. These contact sites are highly dynamic and establish an interconnected organellar network able to quickly respond to external and internal stress by changing size, abundance and molecular architecture. Here, we discuss recent work on nucleus-vacuole junctions, connecting yeast vacuoles with the nucleus. Appearing as small, single foci in mitotic cells, these contacts expand into one enlarged patch upon nutrient exhaustion and entry into quiescence or can be shaped into multiple large foci essential to sustain viability upon proteostatic stress at the nuclear envelope. We highlight the remarkable plasticity and rapid remodelling of these contact sites upon metabolic or proteostatic stress and their emerging importance for cellular fitness.

Snd3 controls nucleus-vacuole junctions in response to glucose signaling.

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance.

Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.

Filling the Antibody Pipeline in Allergy: PIPE Cloning of IgE, IgG1 and IgG4 against the Major Birch Pollen Allergen Bet v 1.

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG1, and IgG4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.

Hsp70-mediated quality control: should I stay or should I go?

Chaperones of the 70 kDa heat shock protein (Hsp70) superfamily are key components of the cellular proteostasis system. Together with its co-chaperones, Hsp70 forms proteostasis subsystems that antagonize protein damage during physiological and stress conditions. This function stems from highly regulated binding and release cycles of protein substrates, which results in a flow of unfolded, partially folded and misfolded species through the Hsp70 subsystem. Specific factors control how Hsp70 makes decisions regarding folding and degradation fates of the substrate proteins. In this review, we summarize how the flow of Hsp70 substrates is controlled in the cell with special emphasis on recent advances regarding substrate release mechanisms.

Closing the Gap: Membrane Contact Sites in the Regulation of Autophagy.

In all eukaryotic cells, intracellular organization and spatial separation of incompatible biochemical processes is established by individual cellular subcompartments in form of membrane-bound organelles. Virtually all of these organelles are physically connected via membrane contact sites (MCS), allowing interorganellar communication and a functional integration of cellular processes. These MCS coordinate the exchange of diverse metabolites and serve as hubs for lipid synthesis and trafficking. While this of course indirectly impacts on a plethora of biological functions, including autophagy, accumulating evidence shows that MCS can also directly regulate autophagic processes. Here, we focus on the nexus between interorganellar contacts and autophagy in yeast and mammalian cells, highlighting similarities and differences. We discuss MCS connecting the ER to mitochondria or the plasma membrane, crucial for early steps of both selective and non-selective autophagy, the yeast-specific nuclear-vacuolar tethering system and its role in microautophagy, the emerging function of distinct autophagy-related proteins in organellar tethering as well as novel MCS transiently emanating from the growing phagophore and mature autophagosome.

Capacitive coupling increases the accuracy of cell-specific tumour disruption by electric fields.

Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations.

Apitoxin and Its Components against Cancer, Neurodegeneration and Rheumatoid Arthritis: Limitations and Possibilities.

Natural products represent important sources for the discovery and design of novel drugs. Bee venom and its isolated components have been intensively studied with respect to their potential to counteract or ameliorate diverse human diseases. Despite extensive research and significant advances in recent years, multifactorial diseases such as cancer, rheumatoid arthritis and neurodegenerative diseases remain major healthcare issues at present. Although pure bee venom, apitoxin, is mostly described to mediate anti-inflammatory, anti-arthritic and neuroprotective effects, its primary component melittin may represent an anticancer therapeutic. In this review, we approach the possibilities and limitations of apitoxin and its components in the treatment of these multifactorial diseases. We further discuss the observed unspecific cytotoxicity of melittin that strongly restricts its therapeutic use and review interesting possibilities of a beneficial use by selectively targeting melittin to cancer cells.