Cellular Quality Control System Research Articles

14-3-3τ as a Modulator of Early α-Synuclein Multimerization and Amyloid Formation.

The aggregation of α-synuclein (αS) plays a key role in Parkinson's disease (PD) etiology. While the onset of PD is age-related, the cellular quality control system appears to regulate αS aggregation throughout most human life. Intriguingly, the protein 14-3-3τ has been demonstrated to delay αS aggregation and the onset of PD in various models. However, the molecular mechanisms behind this delay remain elusive. Our study confirms the delay in αS aggregation by 14-3-3τ, unveiling a concentration-dependent relation. Utilizing microscale thermophoresis (MST) and single-molecule burst analysis, we quantified the early αS multimers and concluded that these multimers exhibit properties that classify them as nanoscale condensates that form in a cooperative process, preceding the critical nucleus for fibril formation. Significantly, the αS multimer formation mechanism changes dramatically in the presence of scaffold protein 14-3-3τ. Our data modeling suggests that 14-3-3τ modulates the multimerization process, leading to the creation of mixed multimers or co-condensates, comprising both αS and 14-3-3τ. These mixed multimers form in a noncooperative process. They are smaller, more numerous, and distinctively not on the pathway to amyloid formation. Importantly, 14-3-3τ thus acts in the very early stage of αS multimerization, ensuring that αS does not aggregate but remains soluble and functional. This offers long-sought novel entries for the pharmacological modulation of PD.

Therapeutic Evaluation of Thermal Heat Shock Response in Mouse Model of Alzheimer’s disease

AbstractBackgroundThe accumulation of toxic amyloid‐ß protein in brain is a hallmark of Alzheimer’s disease. The accumulation of such toxicants is prevented by a cellular protein quality control system. Molecular chaperons, which further prevent proteins from misfolding and aggregating, are produced by this system, which is mostly activated in response to acute thermal stress. This study aims to determine the therapeutic effect of frequent thermal shock on the development of Alzheimer’s diseaseMethodThe induction of Alzheimer’s disease was caused by the intracerebroventricular (ICV) injection of the Aß (1‐42) peptide into the ventricular system of the mouse brain. The animals were divided into four groups: normal control (NC), disease control (DC), sham control (SC), water immersion/no heat group (NH), and hot water immersion/high heat group (HH). The ICV‐Aß injection was given to the DC, NH, and HH groups to induce dementia and AD‐like symptoms. The animals in the HH treatment group were given heat shock in the form of hot water immersion for a period of 4 weeks, where each session lasted for 20 minutes. The animals in the NH group were also exposed to water immersion for 20 minutes daily for a period of 4 weeks, but the water temperature was maintained at room temperature. After the completion of the treatment period, the behavioural and biochemical parameters of the animals were assessed for learning and memory defects. The enzyme‐linked immunosorbent assay (ELISA) was carried out for the Aß estimation.ResultBehavioural assessments in the Y‐maze and morris water maze showed that the animals given daily heat exposure for a period of 4 weeks resisted the detrimental memory effects of the ICV‐Aß injection and had better memory than the water immersion group with no heat treatment given. Increased levels of lipid peroxidation and decreased levels of glutathione were significantly (p<0.05) antagonised by hot water immersion treatment. ELISA showed the lower level of Aß in hot water immersion/high heat group animals when compared to the disease control animals.ConclusionThe study concluded that frequent heat exposure from hot baths and saunas may reduce the risk of developing dementia and Alzheimer’s‐like symptoms in old age.

Lipid Quality Control and Ferroptosis: From Concept to Mechanism.

Cellular quality control systems sense and mediate homeostatic responses to prevent the buildup of aberrant macromolecules, which arise from errors during biosynthesis, damage by environmental insults, or imbalances in enzymatic and metabolic activity. Lipids are structurally diverse macromolecules that have many important cellular functions, ranging from structural roles in membranes to functions as signaling and energy-storage molecules. As with other macromolecules, lipids can be damaged (e.g., oxidized), and cells require quality control systems to ensure that nonfunctional and potentially toxic lipids do not accumulate. Ferroptosis is a form of cell death that results from the failure of lipid quality control and the consequent accumulation of oxidatively damaged phospholipids. In this review, we describe a framework for lipid quality control, using ferroptosis as an illustrative example to highlight concepts related to lipid damage, membrane remodeling, and suppression or detoxification of lipid damage via preemptive and damage-repair lipid quality control pathways. Expected final online publication date for the Annual Review of Biochemistry , Volume 93 is June 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Altered Cellular Protein Quality Control System Modulates Cardiomyocyte Function in Volume Overload-Induced Hypertrophy.

Volume-induced hypertrophy is one of the risk factors for cardiac morbidity and mortality. In addition, mechanical and metabolic dysfunction, aging, and cellular redox balance are also contributing factors to the disease progression. In this study, we used volume overload (VO), which was induced by an aortocaval fistula in 2-month-old male Wistar rats, and sham-operated animals served as control. Functional parameters were measured by transthoracic echocardiography at termination 4- or 8-months after VO. The animals showed hypertrophic remodeling that was accompanied by mechanical dysfunction and increased cardiomyocyte stiffness. These alterations were reversible upon treatment with glutathione. Cardiomyocyte dysfunction was associated with elevated oxidative stress markers with unchanged inflammatory signaling pathways. In addition, we observed altered phosphorylation status of small heat shock proteins 27 and 70 and diminished protease expression caspases 3 compared to the matched control group, indicating an impaired protein quality control system. Such alterations might be attributed to the increased oxidative stress as anticipated from the enhanced titin oxidation, ubiquitination, and the elevation in oxidative stress markers. Our study showed an early pathological response to VO, which manifests in cardiomyocyte mechanical dysfunction and dysregulated signaling pathways associated with enhanced oxidative stress and an impaired protein quality control system.

Dynamic quality control machinery that operates across compartmental borders mediates the degradation of mammalian nuclear membrane proteins.

Many human diseases are caused by mutations in nuclear envelope (NE) proteins. How protein homeostasis and disease etiology are interconnected at the NE is poorly understood. Specifically, the identity of local ubiquitin ligases that facilitate ubiquitin-proteasome-dependent NE protein turnover is presently unknown. Here, we employ a short-lived, Lamin B receptor disease variant as a model substrate in a genetic screen to uncover key elements of NE protein turnover. We identify the ubiquitin-conjugating enzymes (E2s) Ube2G2 and Ube2D3, the membrane-resident ubiquitin ligases (E3s) RNF5 and HRD1, and the poorly understood protein TMEM33. RNF5, but not HRD1, requires TMEM33 both for efficient biosynthesis and function. Once synthesized, RNF5 responds dynamically to increased substrate levels at the NE by departing from the endoplasmic reticulum, where HRD1 remains confined. Thus, mammalian protein quality control machinery partitions between distinct cellular compartments to address locally changing substrate loads, establishing a robust cellular quality control system.

ATP-Dependent Lon Proteases in the Cellular Protein Quality Control System

ATP-Dependent Lon Proteases in the Cellular Protein Quality Control System

The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones.

Protein aggregation, or the uncontrolled self-assembly of partially folded proteins, is an ever-present danger for living organisms. Unimpeded, protein aggregation can result in severe cellular dysfunction and disease. A group of proteins known as molecular chaperones is responsible for dismantling protein aggregates. However, how protein aggregates are recognized and disassembled remains poorly understood. Here we employ a single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS), in combination with two structurally distinct aggregate types grown from the same starting protein, to examine the mechanism of chaperone-mediated protein disaggregation. Using the core bi-chaperone disaggregase system from Escherichia coli as a model, we demonstrate that, in contrast to prevailing models, the overall size of an aggregate particle has, at most, a minor influence on the progression of aggregate disassembly. Rather, we show that changes in internal structure, which have no observable impact on aggregate particle size or molecular chaperone binding, can dramatically limit the ability of the bi-chaperone system to take aggregates apart. In addition, these structural alterations progress with surprising speed, rendering aggregates resistant to disassembly within minutes. Thus, while protein aggregate structure is generally poorly defined and is often obscured by heterogeneous and complex particle distributions, it can have a determinative impact on the ability of cellular quality control systems to process protein aggregates.

Folding state as the Decision Maker in Detecting Misfolded Proteins by Hsp70

Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C‐terminus of Hsc70‐interacting protein (CHIP). Hsp70 itself helps misfolded “client” proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP‐mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above‐mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate‐binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide‐locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.

Proteinopathies as Hallmarks of Impaired Gene Expression, Proteostasis and Mitochondrial Function in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. As with the majority of neurodegenerative diseases, the pathological hallmarks of ALS involve proteinopathies which lead to the formation of various polyubiquitylated protein aggregates in neurons and glia. ALS is a highly heterogeneous disease, with both familial and sporadic forms arising from the convergence of multiple disease mechanisms, many of which remain elusive. There has been considerable research effort invested into exploring these disease mechanisms and in recent years dysregulation of RNA metabolism and mitochondrial function have emerged as of crucial importance to the onset and development of ALS proteinopathies. Widespread alterations of the RNA metabolism and post-translational processing of proteins lead to the disruption of multiple biological pathways. Abnormal mitochondrial structure, impaired ATP production, dysregulation of energy metabolism and calcium homeostasis as well as apoptosis have been implicated in the neurodegenerative process. Dysfunctional mitochondria further accumulate in ALS motor neurons and reflect a wider failure of cellular quality control systems, including mitophagy and other autophagic processes. Here, we review the evidence for RNA and mitochondrial dysfunction as some of the earliest critical pathophysiological events leading to the development of ALS proteinopathies, explore their relative pathological contributions and their points of convergence with other key disease mechanisms. This review will focus primarily on mutations in genes causing four major types of ALS (C9ORF72, SOD1, TARDBP/TDP-43, and FUS) and in protein homeostasis genes (SQSTM1, OPTN, VCP, and UBQLN2) as well as sporadic forms of the disease. Finally, we will look to the future of ALS research and how an improved understanding of central mechanisms underpinning proteinopathies might inform research directions and have implications for the development of novel therapeutic approaches.

An ALS-associated variant of the autophagy receptor SQSTM1/p62 reprograms binding selectivity toward the autophagy-related hATG8 proteins

Recognition of human autophagy-related 8 (hATG8) proteins by autophagy receptors represents a critical step within this cellular quality control system. Autophagy impairment is known to be a pathogenic mechanism in the motor neuron disorder amyotrophic lateral sclerosis (ALS). Overlapping but specific roles of hATG8 proteins belonging to the LC3 and GABARAP subfamilies are incompletely understood, and binding selectivity is typically overlooked. We previously showed that an ALS-associated variant of the SQSTM1/p62 (p62) autophagy receptor bearing an L341V mutation within its ATG8-interacting motif (AIM) impairs recognition of LC3B in vitro, yielding an autophagy-deficient phenotype. Improvements in understanding of hATG8 recognition by AIMs now distinguish LC3-interaction and GABARAP-interaction motifs and predict the effects of L341V substitution may extend beyond loss of function to biasing AIM binding preference. Through biophysical analyses, we confirm impaired binding of the L341V-AIM mutant to LC3A, LC3B, GABARAP, and GABARAPL1. In contrast, p62 AIM interactions with LC3C and GABARAPL2 are unaffected by this mutation. Isothermal titration calorimetry and NMR investigations provided insights into the entropy-driven GABARAPL2/p62 interaction and how the L341V mutation may be tolerated. Competition binding demonstrated reduced association of the L341V-AIM with one hATG8 manifests as a relative increase in association with alternate hATG8s, indicating effective reprogramming of hATG8 selectivity. These data highlight how a single AIM peptide might compete for binding with different hATG8s and suggest that the L341V-AIM mutation may be neomorphic, representative of a disease mechanism that likely extends into other human disorders.

Autophagy-Lysosomal Pathway as Potential Therapeutic Target in Parkinson's Disease.

Cellular quality control systems have gained much attention in recent decades. Among these, autophagy is a natural self-preservation mechanism that continuously eliminates toxic cellular components and acts as an anti-ageing process. It is vital for cell survival and to preserve homeostasis. Several cell-type-dependent canonical or non-canonical autophagy pathways have been reported showing varying degrees of selectivity with regard to the substrates targeted. Here, we provide an updated review of the autophagy machinery and discuss the role of various forms of autophagy in neurodegenerative diseases, with a particular focus on Parkinson’s disease. We describe recent findings that have led to the proposal of therapeutic strategies targeting autophagy to alter the course of Parkinson’s disease progression.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

The deubiquitinase USP11 is a versatile and conserved regulator of autophagy

Autophagy is a major cellular quality control system responsible for the degradation of proteins and organelles in response to stress and damage to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase ubiquitin-specific protease 11 (USP11) restricts autophagy and that KO of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human amyloid-β peptide 42 aggregation-induced paralysis. USP11 coprecipitated with autophagy-specific class III phosphatidylinositol 3-kinase complex I and limited its interaction with nuclear receptor-binding factor 2, thus decreasing lipid kinase activity of class III phosphatidylinositol 3-kinase complex I and subsequent recruitment of effectors such as WD-repeat domain phosphoinositide-interacting proteins to the autophagosomal membrane. Accordingly, more WD-repeat domain phosphoinositide-interacting protein 2 puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mechanistic target of rapamycin, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.

Circulating extracellular vesicles: friends and foes in neurodegeneration.

Extracellular vesicles have been identified as pivotal mediators of intercellular communication with critical roles in physiological and pathological conditions. Via this route, several molecules (e.g., nucleic acids, proteins, metabolites) can be transferred to proximal and distant targets to convey specific information. Extracellular vesicle-associated cargo molecules have been proposed as markers of several disease conditions for their potential of tracking down the generating cell. Indeed, circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems especially in conditions characterized by the accrual of intracellular misfolded proteins. Furthermore, the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo molecules to diseased tissues makes these circulating shuttles possible targets for therapeutic development. The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-associated proteins might reflect molecular events occurring in hardly accessible tissues, such as the brain, thereby serving as a “brain liquid biopsy”. The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches. Here, we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases (i.e., Parkinson’s and Alzheimer’s diseases). A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.

The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.

Alzheimer’s Disease (AD) is the most common form of dementia, characterised by intra- and extracellular protein aggregation. In AD, the cellular protein quality control (PQC) system is derailed and fails to prevent the formation of these aggregates. Especially the mitochondrial paralogue of the conserved Hsp90 chaperone class, tumour necrosis factor receptor-associated protein 1 (TRAP1), is strongly downregulated in AD, more than other major PQC factors. Here, we review molecular mechanism and cellular function of TRAP1 and subsequently discuss possible links to AD. TRAP1 is an interesting paradigm for the Hsp90 family, as it chaperones proteins with vital cellular function, despite not being regulated by any of the co-chaperones that drive its cytosolic paralogues. TRAP1 encloses late folding intermediates in a non-active state. Thereby, it is involved in the assembly of the electron transport chain, and it favours the switch from oxidative phosphorylation to glycolysis. Another key function is that it ensures mitochondrial integrity by regulating the mitochondrial pore opening through Cyclophilin D. While it is still unclear whether TRAP1 itself is a driver or a passenger in AD, it might be a guide to identify key factors initiating neurodegeneration.

Breast milk MSCs upregulated β-cells PDX1, Ngn3, and PCNA expression via remodeling ER stress /inflammatory /apoptotic signaling pathways in type 1 diabetic rats

Type 1 diabetes mellitus (T1DM) is one of the autoimmune diseases characterized by beta-cell dysfunction with serious health complications. Br-MSCs represent a novel valid candidate in regenerative medicine disciplines. Yet, the full potential of Br-MSCs in managing type 1 diabetes remains elusive. Indeed, this study was designed to explore a novel approach investigating the possible regenerative capacity of Br-MSCs in type1 diabetic islet on the level of the cellular mRNA expression of different molecular pathways involved in pancreatic beta-cell dysfunction. Sixty adult male Sprague-Dawley rats were randomly assigned into 3 groups (20 rats each); the control group, type1 diabetic group, and the type 1 diabetic Br-MSCs treated group. And, for the first time, our results revealed that intraperitoneally transplanted Br-MSCs homed to the diabetic islet and improved fasting blood glucose, serum insulin level, pancreatic oxidative stress, upregulated pancreatic mRNA expression for: regenerative markers (Pdx1, Ngn3, PCNA), INS, beta-cell receptors (IRS1, IRβ, PPARγ), pancreatic growth factors (IGF-1, VEGFβ1, FGFβ), anti-inflammatory cytokine (IL10) and anti-apoptotic marker (BCL2) too, Br-MSCs downregulated pancreatic mRNA expression for: inflammatory markers (NFKβ, TNFα, IL1β, IL6, IL8, MCP1), apoptotic markers for both intrinsic and extrinsic pathways (FAS, FAS-L, P53, P38, BAX, Caspase3), ER stress markers (ATF6, ATF3, ATF4, BIP, CHOP, JNK, XBP1) and autophagy inhibitor (mTOR). In conclusion, Br-MSCs could be considered as a new insight in beta cell regenerative therapy improving the deteriorated diabetic islet microenvironment via modulating; ER stress, inflammatory, and apoptotic signaling pathways besides, switching on the cellular quality control system (autophagy) thus enhancing beta-cell function.

Salvage NAD+ biosynthetic pathway enzymes moonlight as molecular chaperones to protect against proteotoxicity.

Human neurodegenerative proteinopathies are disorders associated with abnormal protein depositions in brain neurons. They include polyglutamine (polyQ) conditions such as Huntington's disease (HD) and α-synucleinopathies such as Parkinson's disease (PD). Overexpression of NMNAT/Nma1, an enzyme in the NAD+ biosynthetic salvage pathway, acts as an efficient suppressor of proteotoxicities in yeast, fly and mouse models. Screens in yeast models of HD and PD allowed us to identify three additional enzymes of the same pathway that achieve similar protection against proteotoxic stress: Npt1, Pnc1 and Qns1. The mechanism by which these proteins maintain proteostasis has not been identified. Here, we report that their ability to maintain proteostasis in yeast models of HD and PD is independent of their catalytic activity and does not require cellular protein quality control systems such as the proteasome or autophagy. Furthermore, we show that, under proteotoxic stress, the four proteins are recruited as molecular chaperones with holdase and foldase activities. The NAD+ salvage proteins act by preventing misfolding and, together with the Hsp90 chaperone, promoting the refolding of extended polyQ domains and α-synuclein (α-Syn). Our results illustrate the existence of an evolutionarily conserved strategy of repurposing or moonlighting housekeeping enzymes under stress conditions to maintain proteostasis. We conclude that the entire salvage NAD+ biosynthetic pathway links NAD+ metabolism and proteostasis and emerges as a target for therapeutics to combat age-associated neurodegenerative proteotoxicities.

Pharmacological chaperone action in humanized mouse models of MC4R-linked obesity.

MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PCs) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor–linked (MC4R-linked) obesity. To test the therapeutic PC potential, we engineered humanized MC4R (hMC4R) mouse models expressing either the WT human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof of principle for the therapeutic potential of MC4R-targeting PCs in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to α–melanocyte-stimulating hormone (α-MSH) but not β-MSH or melanotan II, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created 2 new obesity models, a hypomorphic highlighting species differences and an amorphic providing a preclinical model to test the therapeutic potential of PCs to treat MC4R-linked obesity.

The alarmones (p)ppGpp are part of the heat shock response of Bacillus subtilis.

Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.

Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio‐orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence‐based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse‐chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.