Peroxisome formation and maintenance are dependent on the endoplasmic reticulum.

Looks can be deceiving. Although peroxisomes appear to be simple organelles, their formation and maintenance pose unique challenges for the cell. The birth of new peroxisomes starts at the endoplasmic reticulum (ER), which delivers lipids and membrane proteins. To form a new peroxisomal compartment, ER-derived preperoxisomal vesicles carrying different membrane proteins fuse, allowing the assembly of the peroxisomal translocon. To complete formation, peroxisomes import their soluble proteins directly from the cytosol using the newly assembled translocon. Together with the ER-derived biogenic route, peroxisomal fission and segregation subsequently maintain the cellular peroxisome population. In this review we highlight the latest insights on the life cycle of peroxisomes and show how the new cell biology concept of peroxisome formation affects our thinking about peroxisome-related diseases and their evolutionary past. The future challenge lies in the identification of all the proteins involved in this elaborate biogenic process and the dissection of their mechanism of action.

Biochemically distinct vesicles from the endoplasmic reticulum fuse to form peroxisomes.

As a rule, organelles in eukaryotic cells can derive only from pre-existing organelles. Peroxisomes are unique because they acquire their lipids and membrane proteins from the endoplasmic reticulum (ER), whereas they import their matrix proteins directly from the cytosol. We have discovered that peroxisomes are formed via heterotypic fusion of at least two biochemically distinct preperoxisomal vesicle pools that arise from the ER. These vesicles each carry half a peroxisomal translocon complex. Their fusion initiates assembly of the full peroxisomal translocon and subsequent uptake of enzymes from the cytosol. Our findings demonstrate a remarkable mechanism to maintain biochemical identity of organelles by transporting crucial components via different routes to their final destination.

Protein folding and modification in the mammalian endoplasmic reticulum.

Analysis of the human genome reveals that approximately a third of all open reading frames code for proteins that enter the endoplasmic reticulum (ER), demonstrating the importance of this organelle for global protein maturation. The path taken by a polypeptide through the secretory pathway starts with its translocation across or into the ER membrane. It then must fold and be modified correctly in the ER before being transported via the Golgi apparatus to the cell surface or another destination. Being physically segregated from the cytosol means that the ER lumen has a distinct folding environment. It contains much of the machinery for fulfilling the task of protein production, including complex pathways for folding, assembly, modification, quality control, and recycling. Importantly, the compartmentalization means that several modifications that do not occur in the cytosol, such as glycosylation and extensive disulfide bond formation, can occur to secreted proteins to enhance their stability before their exposure to the extracellular milieu. How these various machineries interact during the normal pathway of folding and protein secretion is the subject of this review.

Hold the fold: how delayed folding aids protein secretion.

In bacteria, N-terminal signal peptides mark proteins for transport across the plasma membrane. A recent study by Smets et al (2022) followed the folding of a pair of structural twins to shed light on how evolution has optimised the secretory process.

ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway.

The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.

Transmembrane Helices 7 and 8 Confer Aggregation Sensitivity to the Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability.

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue.

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Redefining Hypo- and Hyper-Responding Phenotypes of CFTR Mutants for Understanding and Therapy.

Mutations in CFTR cause misfolding and decreased or absent ion-channel function, resulting in the disease Cystic Fibrosis. Fortunately, a triple-modulator combination therapy (Trikafta) has been FDA-approved for 178 mutations, including all patients who have F508del on one allele. That so many CFTR mutants respond well to modulators developed for a single mutation is due to the nature of the folding process of this multidomain protein. We have addressed the question 'What characterizes the exceptions: the mutants that functionally respond either not or extremely well'. A functional response is the product of the number of CFTR molecules on the cell surface, open probability, and conductivity of the CFTR chloride channel. By combining biosynthetic radiolabeling with protease-susceptibility assays, we have followed CF-causing mutants during the early and late stages of folding in the presence and absence of modulators. Most CFTR mutants showed typical biochemical responses for each modulator, such as a TMD1 conformational change or an increase in (cell-surface) stability, regardless of a functional response. These modulators thus should still be considered for hypo-responder genotypes. Understanding both biochemical and functional phenotypes of outlier mutations will boost our insights into CFTR folding and misfolding, and lead to improved therapeutic strategies.

Clinical and molecular characterization of the R751L-CFTR mutation.

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly, inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.

ERdj5 is the ER reductase that catalyzes the removal of non-native disulfides and correct folding of the LDL receptor.

ERdj5 is a member of the protein disulfide isomerase family of proteins localized to the endoplasmic reticulum (ER) of mammalian cells. To date, only a limited number of substrates for ERdj5 are known. Here we identify a number of endogenous substrates that form mixed disulfides with ERdj5, greatly expanding its client repertoire. ERdj5 previously had been thought to exclusively reduce disulfides in proteins destined for dislocation to the cytosol for degradation. However, we demonstrate here that for one of the identified substrates, the low-density lipoprotein receptor (LDLR), ERdj5 is required not for degradation, but rather for efficient folding. Our results demonstrate that the crucial role of ERdj5 is to reduce non-native disulfides formed during productive folding and that this requirement is dependent on its interaction with BiP. Hence, ERdj5 acts as the ER reductase, both preparing misfolded proteins for degradation and catalyzing the folding of proteins that form obligatory non-native disulfides.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

Alteration of protein function by a silent polymorphism linked to tRNA abundance.

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

Co- and Post-Translational Protein Folding in the ER.

The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit - if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding.

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules.

The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.

Mutational and functional analysis of N-linked glycosylation of envelope fusion protein F of Helicoverpa armigera nucleopolyhedrovirus.

The envelope fusion (F) protein of baculoviruses is a heavily N-glycosylated protein that plays a significant role in the virus infection cycle. N-Linked glycosylation of virus envelope glycoprotein is important for virus envelope glycoprotein folding and its function in general. There are six predicted N-glycosylation sites in the F (HaF) protein of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The N-glycosylation site located in the F(2) subunit (N104) of HaF has been identified and functionally characterized previously (Long et al., 2007). In this study, the other five potential N-glycosylation sites located in the HaF1 subunit, namely, N293, N361, N526, N571 and N595, were analysed extensively to examine their N-glycosylation and relative importance to the function of HaF. The results showed that four of these five potential glycosylation sites in the F(1) subunit, N293, N361, N526 and N571, were N-glycosylated in F proteins of mature HearNPV budded viruses (BVs) but that N595 was not. In general, the conserved site N526 was critical to the functioning of HaF, as absence of N-glycosylation of N526 reduced the efficiency of HaF folding and trafficking, consequently decreased fusogenicity and modified the subcellular localization of HaF proteins, and thus impaired virus production and infectivity. The absence of N-glycosylation at other individual sites was found to have different effects on the fusogenicity and subcelluar distribution of HaF proteins in HzAM1 cells. In summary, N-glycosylation plays comprehensive roles in HaF function and virus infectivity, which is further discussed.

Peroxisomes: minted by the ER.

Peroxisomes are one of numerous organelles in a eukaryotic cell; they are small, single-membrane-bound vesicles involved in cellular metabolism, particularly fatty acid degradation. Transport of metabolites and co-factors in and across the membrane is taken care of by specific transporters. Peroxisome formation and maintenance has been debated for a long time: opinions swinging from autonomous to ER-derived organelles. Only recently it has been established firmly that the site of origin of peroxisomes is the ER. It implies that a new branch of the endomembrane system is open to further characterization.

The Folding Pathway of ABC Transporter CFTR: Effective and Robust.

De novo protein folding into a native three-dimensional structure is indispensable for biological function, is instructed by its amino acid sequence, and occurs along a vectorial trajectory. The human proteome contains thousands of membrane-spanning proteins, whose biosynthesis begins on endoplasmic reticulum-associated ribosomes. Nearly half of all membrane proteins traverse the membrane more than once, including therapeutically important protein families such as solute carriers, G-protein-coupled receptors, and ABC transporters. These mediate a variety of functions like signal transduction and solute transport and are often of vital importance for cell function and tissue homeostasis. Missense mutations in multispan membrane proteins can lead to misfolding and cause disease; an example is the ABC transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Even though our understanding of multispan membrane-protein folding still is rather rudimental, the cumulative knowledge of 20 years of basic research on CFTR folding has led to development of drugs that modulate the misfolded protein. This has provided the prospect of a life without CF to the vast majority of patients. In this review we describe our understanding of the folding pathway of CFTR in cells, which is modular and tolerates many defects, making it effective and robust. We address how modulator drugs affect folding and function of CFTR, and distinguish protein stability from its folding process. Since the domain architecture of (mammalian) ABC transporters are highly conserved, we anticipate that the insights we discuss here for folding of CFTR may lay the groundwork for understanding the general rules of ABC-transporter folding.

Structure basis of CFTR folding, function and pharmacology.

The root cause of cystic fibrosis (CF), the most common life-shortening genetic disease in the Caucasian population, is the loss of function of the CFTR protein, which serves as a phosphorylation-activated, ATP-gated anion channel in numerous epithelia-lining tissues. In the past decade, high-throughput drug screening has made a significant stride in developing highly effective CFTR modulators for the treatment of CF. Meanwhile, structural-biology studies have succeeded in solving the high-resolution three-dimensional (3D) structure of CFTR in different conformations. Here, we provide a brief overview of some striking features of CFTR folding, function and pharmacology, in light of its specific structural features within the ABC-transporter superfamily. A particular focus is given to CFTR's first nucleotide-binding domain (NBD1), because folding of NBD1 constitutes a bottleneck in the CFTR protein biogenesis pathway, and ATP binding to this domain plays a unique role in the functional stability of CFTR. Unraveling the molecular basis of CFTR folding, function, and pharmacology would inspire the development of next-generation mutation-specific CFTR modulators.