Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite

A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium.Malaria is a major healthcare problem worldwide resulting in an estimated 0.65 million deaths every year. Present strategy of malaria control is totally dependent on pharmacological treatments and there is a constant need to identify new drug targets involved in important metabolic pathways in the parasite.¹ The cellular machinery responsible for protein quality control and folding is essential for cellular homeostasis and survival of eukaryotic cells. The protein quality control is particularly important for malaria parasites because of its high replication rate, high temperature stress and high load on endoplasmic reticulum (ER) because of large amount of proteins that are to be secreted or exported to the host cytosol. In eukaryotic cells, inhibition of 26 S proteasome is one of the major causes for low clearance of unfolded proteins from ER and therefore leads to ER stress. ER stress response may help the cell to survive through the stress, it can also trigger apoptosis when high levels of unfolded proteins persist for a longer time.² We have earlier shown that disruption of an important metabolic pathway of the parasite can incite the parasite to undergo apoptosis-like cell death.³ A number of other studies have suggested that apoptosis-like cell death can be induced in Plasmodium falciparum by different anti-malarial drugs, antibiotics and other small molecules.^{4, 5} However, the mode of induction of cell death and different cascade of molecular/cellular events leading to apoptosis-like cell death in the parasite are not clearly understood.In this study, we have assessed cellular stress induced by proteasome inhibition on asexual stage P. falciparum parasites. Global quantitative proteomic analyses identified putative pro-survival pathways in the parasites under cellular stress. We further show that persistent proteasome inhibition cause parasite cell death, which is mediated by a cascade of molecular and cellular events. Overall, our results highlight a probable mechanism of cell death and survival in Plasmodium under cellular stress.     

Post-translocational folding of secretory proteins in Gram-positive bacteria

The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.     

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.     

内质网应激响应在脂肪组织中的代谢调节作用

在真核细胞中,内质网是蛋白合成、加工及质量监控的关键细胞器,也是Ca2+储存及脂质合成的重要场所.细胞通过未折叠蛋白响应(unfolded protein response,UPR)感应外界不同刺激引发的内质网应激,在维持细胞功能稳态中发挥至关重要的作用.在哺乳动物中,三个位于内质网的跨膜蛋白——肌醇依赖酶la(ino...     

Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.     

Endoplasmic reticulum stress and lipids in health and diseases

The endoplasmic reticulum (ER) is a complex and dynamic organelle that regulates many cellular pathways, including protein synthesis, protein quality control, and lipid synthesis. When one or multiple ER roles are dysregulated and saturated, the ER enters a stress state, which, in turn, activates the highly conserved unfolded protein response (UPR). By sensing the accumulation of unfolded proteins or lipid bilayer stress (LBS) at the ER, the UPR triggers pathways to restore ER homeostasis and eventually induces apoptosis if the stress remains unresolved. In recent years, it has emerged that the UPR works intimately with other cellular pathways to maintain lipid homeostasis at the ER, and so does at cellular levels. Lipid distribution, along with lipid anabolism and catabolism, are tightly regulated, in part, by the ER. Dysfunctional and overwhelmed lipid-related pathways, independently or in combination with ER stress, can have reciprocal effects on other cellular functions, contributing to the development of diseases. In this review, we summarize the current understanding of the UPR in response to proteotoxic stress and LBS and the breadth of the functions mitigated by the UPR in different tissues and in the context of diseases.     

细胞内质网应激与糖脂代谢紊乱的机制关联

在真核细胞中,内质网是蛋白质合成、折叠、加工及其质量监控的重要场所。当内质网难以承担蛋白折叠的高负荷时则引发内质网应激(ER stress),激活细胞的未折叠蛋白响应(unfoldedprotein response,UPR)。细胞通过内质网跨膜蛋白ATF6、PERK和IRE1介导的三条极为关键的UPR信号通路,调控下游相关基因的表达,以增强内质网对蛋白折叠的处理能力。因此,UPR通路在细胞的稳态平衡中具有举足轻重的作用,而这一动态过程的调控对于维持机体的正常生理功能至关重要。近来大量研究表明,在哺乳动物中内质网应激与机体的营养感应和糖脂代谢的调控过程密切相关。在肝脏、脂肪、胰岛以及下丘脑等不同的组织器官中,内质网应激均影响代谢通路的调节机制,因此在糖脂代谢紊乱的发生发展中扮演重要的角色。综上所述,进一步深入了解内质网应激引发代谢异常的生理学机制,可以为肥胖、脂肪肝及2型糖尿病等相关代谢性疾病的防治提供新的潜在药物靶点和重要的理论线索。     

Size‐dependent secretory protein reflux into the cytosol in association with acute endoplasmic reticulum stress

Once secretory proteins have been targeted to the endoplasmic reticulum (ER) lumen, the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER‐Associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process “ER reflux.” Reflux appears to be regulated in a size‐dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress‐induced reflux. Together, our findings establish a new ER stress‐regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and preemptive quality control pathways. Importantly, our results highlight the value of fully characterizing the cell biology of reporters and describe a simple modification to maintain luminal ER reporters in the ER during acute ER stress.     

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

Repression of Orf2971 induces accumulation of chloroplast precursor proteins and impaired chloroplast quality indicating that Orf2971 is required for protein import and chloroplast quality control.

IN A NUTSHELL Background: The chloroplast is an important bioreactor as well as a photosynthetic site. Approximately 3,000 plastid proteins encoded in the nucleus are translocated into the chloroplast envelope via the TOC (translocon at the outer chloroplast envelope) and TIC machineries. Most nucleus-encoded preproteins that enter the plastid are unfolded as they traverse the TOC–TIC import complexes. To prevent these unfolded or misfolded proteins from causing chloroplast damage, a quality control mechanism comprising molecular chaperones and proteases ensures that all polypeptides entering chloroplasts are either correctly folded or degraded. However, there is still no consensus on the TIC complex’s components, motor proteins, or mechanism for refolding proteins entering the chloroplast. Question: What is the precise function of each of the proteins in the TIC complex? What is the composition of the chloroplast protein import machinery motor? How are the newly imported chloroplast proteins refolded and assembled into functional complexes? Findings: We found that Orf2971, encoded by the largest gene in the Chlamydomonas reinhardtii chloroplast genome and proposed to be an ortholog of Ycf2, is directly associated with the protein import machinery and plays a crucial role in ensuring the quality of proteins targeted to the chloroplast. Orf2971 deficiency induces protein precursor accumulation, partial proteolysis and protein aggregation, increased expression of chaperones and proteases, and autophagy. We hypothesize that Orf2971 is intimately linked to the protein import machinery and plays a critical role in chloroplast protein quality control. Next steps: The next challenge is to identify the sorting components associated with this complex on the stromal side. Furthermore, additional experimental evidence is required to investigate the relationship between different import machineries, including the analysis of the accumulation of precursor proteins in the various import mutants.     

Protein aggregation in Escherichia coli: role of proteases

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.     

A disulfide bond-containing alkaline phosphatase triggers a BdbC-dependent secretion stress response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

Endoplasmic Reticulum Enrollment in Alzheimer’s Disease

Alzheimer??s disease (AD) poses a huge challenge for society and health care worldwide as molecular pathogenesis of the disease is poorly understood and curative treatment does not exist. The mechanisms leading to accelerated neuronal cell death in AD are still largely unknown, but accumulation of misfolded disease-specific proteins has been identified as potentially involved. In the present review, we describe the essential role of endoplasmic reticulum (ER) in AD. Despite the function that mitochondria may play as the central major player in the apoptotic process, accumulating evidence highlights ER as a critical organelle in AD. Stress that impairs ER physiology leads to accumulation of unfolded or misfolded proteins, such as amyloid ?? (A??) peptide, the major component of amyloid plaques. In an attempt to ameliorate the accumulation of unfolded proteins, ER stress triggers a protective cellular mechanism, which includes the unfolded protein response (UPR). However, when activation of the UPR is severe or prolonged enough, the final cellular outcome is pathologic apoptotic cell death. Distinct pathways can be activated in this process, involving stress sensors such as the JNK pathway or ER chaperones such as Bip/GRP94, stress modulators such as Bcl-2 family proteins, or even stress effectors such as caspase-12. Here, we detail the involvement of the ER and associated stress pathways in AD and discuss potential therapeutic strategies targeting ER stress.     

Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ER and tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.     

Differential proteomic profiling identifies novel molecular targets of pterostilbene against experimental diabetes

Pterostilbene (PTS), a naturally occurring stilbene, confers protection against oxidative and cytokine stress induced pancreatic β-cell apoptosis in vitro and in vivo. To provide insights into the molecular mechanism, we performed a proteomic study on the pancreas of PTS-treated diabetic mice using electrospray ionization tandem–mass spectrometry (LC–MS/MS). A total of 1,260 proteins were detected in triplicate samples. Of which, 359 proteins were found to be differentially regulated in streptozotocin-induced diabetic mice pancreas with two fold difference ( P < 0.05, two or more peptides) and on PTS treatment 315 proteins were normalized to control levels. Gene ontology (GO) indicated that majority of the differentially regulated proteins are involved in cellular functions such as metabolism, cellular structure, oxidative stress, endoplasmic-reticulum-associated protein degradation (ERAD) pathway and several stress sensors. Protein–protein interaction network analysis of these differentially expressed proteins showed clustering of proteins involved in protein processing in endoplasmic reticulum (protein synthesis machinery and protein folding), oxidative phosphorylation/oxidative stress proteins, oligosaccharide metabolic process, and antioxidant activity. Our results highlighted that PTS administration rehabilitated the defective metabolic process and redox imbalance, and also suppressed the unfolded protein response and ERAD pathways. The effects on targeting ER machinery and suppressing oxidative stress suggest the great potential of PTS for diabetes management.     

线粒体蛋白质组及蛋白质量控制

线粒体是哺乳动物细胞内重要细胞器,不仅通过氧化磷酸化产生ATP为细胞提供能量,也参与调节钙离子稳态、活性氧(reactive oxygen species,ROS)的产生、细胞应激反应和细胞死亡等过程,其功能障碍不仅导致多种人类疾病的发生,而且也能降低动物卵母细胞质量和早期胚胎发育能力.大量证据表明,线粒体的功能依赖于...     

ERAD substrate recognition in budding yeast

During protein synthesis, the orderly progression of folding, modification, and assembly is paramount to function and vis-à-vis cellular viability. Accordingly, sophisticated quality control mechanisms have evolved to monitor protein maturation throughout the cell. Proteins failing at any step are segregated and degraded as a preventative measure against potential toxicity. Although protein quality control is generally poorly understood, recent research advances in endoplasmic reticulum-associated degradation (ERAD) pathways have provided the most detailed view so far. The discovery of distinct substrate processing sites established a biochemical basis for genetic profiles of model misfolded proteins. Detailed mechanisms for substrate recognition were recently uncovered. For some proteins, sequential glycan trimming steps set a time window for folding. Proteins still unfolded at the final stage expose a specific degradation signal recognized by the ERAD machinery. Through this mechanism, the system does not in fact know that a molecule is “misfolded”. Instead, it goes by the premise that proteins past due have veered off their normal folding pathways and therefore aberrant.