Defective Protein Trafficking Research Articles

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

OXR1 maintains the retromer to delay brain aging under dietary restriction

Dietary restriction (DR) delays aging, but the mechanism remains unclear. We identified polymorphisms in mtd, the fly homolog of OXR1, which influenced lifespan and mtd expression in response to DR. Knockdown in adulthood inhibited DR-mediated lifespan extension in female flies. We found that mtd/OXR1 expression declines with age and it interacts with the retromer, which regulates trafficking of proteins and lipids. Loss of mtd/OXR1 destabilized the retromer, causing improper protein trafficking and endolysosomal defects. Overexpression of retromer genes or pharmacological restabilization with R55 rescued lifespan and neurodegeneration in mtd-deficient flies and endolysosomal defects in fibroblasts from patients with lethal loss-of-function of OXR1 variants. Multi-omic analyses in flies and humans showed that decreased Mtd/OXR1 is associated with aging and neurological diseases. mtd/OXR1 overexpression rescued age-related visual decline and tauopathy in a fly model. Hence, OXR1 plays a conserved role in preserving retromer function and is critical for neuronal health and longevity.

Alcohol-induced tubulin post-translational modifications directly alter hepatic protein trafficking.

Chronic ethanol exposure leads to enhanced protein acetylation and acetaldehyde adduction. Of the multitude of proteins that are modified on ethanol administration, tubulin is among the best studied. However, an open question is whether these modifications are observed in patient samples. Both modifications have also been implicated in promoting alcohol-induced defects in protein trafficking, but whether they do so directly is also unanswered. We first confirmed that tubulin was hyperacetylated and acetaldehyde-adducted in the livers from ethanol-exposed individuals to a similar extent as observed in the livers from ethanol-fed animals and hepatic cells. Livers from individuals with nonalcohol-associated fatty liver showed modest increases in tubulin acetylation, whereas nonalcohol-associated fibrotic human and mouse livers showed virtually no tubulin modifications. We also asked whether tubulin acetylation or acetaldehyde adduction can directly explain the known alcohol-induced defects in protein trafficking. Acetylation was induced by overexpressing the α-tubulin-specific acetyltransferase, αTAT1, whereas adduction was induced by directly adding acetaldehyde to cells. Both αTAT1 overexpression and acetaldehyde treatment significantly impaired plus-end (secretion) and minus-end (transcytosis)-directed microtubule-dependent trafficking and clathrin-mediated endocytosis. Each modification led to similar levels of impairment as observed in ethanol-treated cells. The levels of impairment by either modification showed no dose dependence or no additive effects suggesting that substoichiometric tubulin modifications lead to altered protein trafficking and that lysines are not selectively modified. These results not only confirm that enhanced tubulin acetylation is observed in human livers but that it is most relevant to alcohol-induced injury. Because these tubulin modifications are associated with altered protein trafficking that alters proper hepatic function, we propose that changing the cellular acetylation levels or scavenging free aldehydes are feasible strategies for treating alcohol-associated liver disease.

Biochemical and molecular characterization of the most-common SLC13A5-Epilepsy causing missense-mutations.

The sodium-coupled citrate transporter (NaCT) is a plasma membrane transporter, which is energized by an inwardly directed electrochemical sodium gradient. It mediates the symport of sodium and the carboxylate citrate into cells. NaCT is expressed in the liver, testis, brain, bone, and teeth, where citrate plays key roles in the synthesis of neurotransmitters, cholesterol, and fatty acids, the generation of energy, and teeth/bone mineralization. In humans, loss-of-function mutations in SLC13A5, the NaCT gene, cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5-Epilepsy), which leads to epilepsy, impaired speech, limited motor skills, developmental delay, and tooth defects. Currently, there is no treatment for EIEE25. Recently, the cryo-electron microscopy structure of the human NaCT was solved in an inward-facing conformation. This was an important advancement in the NaCT field, paving the way for a better understanding of the structure-function relationships for this clinically important transporter. We classified 22 NaCT missense disease-causing mutations based on their localizations in the 3D structure. Class I mutations interfere with the transport function by blocking the elevator-type mechanism for substrate translocation. Class II cause defects in protein folding and protein trafficking to the cell surface, which may be corrected by small molecule therapeutics. As there are no NaCT-specific antibodies, we expressed WT and the mutants with specific epitopes to facilitate detection, which didn’t interfere with the presentation of the mutant phenotypes. The Class I mutations C50R, T142M, and T227M displayed protein and surface expression levels similar to WT. Class II mutants G219R, S427L, and L488P showed significantly decreased protein expression and no plasma membrane expression. Both classes displayed diminished transport activity. These experiments have brought us one step closer to understanding the defects of disease-causing mutations at the molecular level, allowing us to begin dissecting NaCT trafficking pathway(s).

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species.

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.

Genetic analysis of and clinical characteristics associated with ANXA11 variants in a Chinese cohort with amyotrophic lateral sclerosis

BackgroundVariants in the annexin A11 gene (ANXA11) have been reported to be associated with amyotrophic lateral sclerosis (ALS). These variants may be involved in the pathogenesis of ALS by causing defects in intracellular protein trafficking. However, the genetic spectrum and clinical characteristics of ALS patients with ANXA11 variants are largely unknown. MethodsGenetic analysis was performed on 1587 Chinese patients with ALS. Eight software packages were used to predict the deleteriousness of missense variants. In addition, we searched PubMed, Embase, and Web of Science for relevant literature and meta-analysed variant frequencies. ResultsIn our ALS cohort, we identified 20 non-synonymous variants in 29 ALS patients, including one stop-gain, one frameshift, and 18 rare missense variants with seven predicted pathogenic variants. In a literature review of 11 reported studies that included 69 patients, 37 ANXA11 variants were reported, with a frequency of 1.7%, which was similar to that in our cohort (1.8%). Both our cohort and previous reports showed that ANXA11 carriers were more commonly males than females (12/17 vs. 19/31). Patients carrying ANXA11 variants affecting the C-terminal of the protein had earlier disease onset and shorter survival times than those carrying variants affecting the N-terminal. We found a relatively longer median survival time than that previously reported (53.6 months vs. 46.0 months). Additionally, Caucasian ANXA11 carriers were more likely to have cognitive impairment, typically frontotemporal dementia (FTD) than their Asian counterparts (20.0% vs. 14.3%). While more than half of the patients in our cohort had cognitive impairment, none had FTD. ConclusionIn our and previously published cases, ALS-associated ANXA11 variants predominantly affected the N- and C-terminal conserved domains. ANXA11 variant carriers are typically male and cognitively impaired. Our study extends the genotypic and phenotypic spectra of ALS patients with ANXA11 variants. Further expansion of the sample size is needed to analyse the clinical and non-motor symptom characteristics of patients and to deepen the understanding of the pathogenesis of ANXA11-associated ALS.

Defective Human SRP Induces Protein Quality Control and Triggers Stress Response

Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

QS6. Defects In Protein Trafficking in Venous Malformations: A Novel Disease Model

QS6. Defects In Protein Trafficking in Venous Malformations: A Novel Disease Model

Molecular physiology of zinc in Drosophila melanogaster

New research in Drosophila melanogaster has revealed the molecular mechanisms of zinc involvement in many biological processes. A newly discovered Metallothionein is predicted to have a higher zinc specificity than the other isoforms. Zinc negatively regulates tyrosine hydroxylase activity by antagonizing iron binding, thus rendering the enzyme ineffective or non-functional. The identification of a new chaperone of the protein disulfide isomerase family provided mechanistic insight into the protein trafficking defects caused by zinc dyshomeostasis in the secretory pathway. Insect models of tumor pathogenesis indicate that zinc regulates the structural stabilization of cells by transcriptionally regulating matrix metalloproteinases while zinc dyshomeostasis in the secretory pathway modulates cell signaling through endoplastic recticulum stress.

Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT–rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.

SPECC1L deficiency causes shortened primary cilia and genetically interacts with IFT‐A deficiency to affect palatogenesis

Orofacial clefts are among the most common congenital malformations, affecting approximately 1 in 700 births. SPECC1L mutations in patients with syndromic cleft lip and/or palate cluster in the second coiled coil domain (CCD2), which facilitates interaction of SPECC1L with microtubules. We have shown that loss of SPECC1L in a null allele (Specc1l∆Ex4) resulted in perinatal lethality and delayed palatal shelf elevation, but no cleft palate (CP) or exencephaly at birth. However, mice with an in‐frame deletion of SPECC1L‐CCD2 exhibited CP and exencephaly, which are common manifestations of ciliopathies. Given that SPECC1L cytoplasmic expression includes localization around the cilia base and that SPECC1L regulates actin cytoskeleton, we investigated an association between SPECC1L and primary cilia. We observed that primary cilia were shortened relative to wild‐type in E13.5 Specc1l∆Ex4/∆Ex4 as well as Specc1l∆CCD2/∆CCD2 mutant palatal mesenchyme and epithelium. The cytoplasmic expression of SPECC1L around the cilia base was diminished in Specc1l∆CCD2/∆CCD2 cells. Thus, loss of SPECC1L association with microtubules appears sufficient to prevent its pericentriolar localization and to cause cilia shortening. Super‐resolution imaging for acetylated‐α‐tubulin and ciliary membrane protein, ARL13B, revealed bulbous structures in Specc1l mutant cilia, suggesting a defect in ciliary protein trafficking. Due to the overlapping phenotypes of shortened cilia, exencephaly and CP between the intraflagellar transport (IFT)‐A mouse mutant, Thm1aln/aln, and Specc1l∆CCD2/∆CCD2, we crossed these mutants to look for a genetic interaction. 18% of Specc1l∆CCD2/+, 0% of Thm1aln/+, and 33% of double heterozygous Specc1l∆CCD2/+;Thm1aln/+ embryos showed CP, confirming a genetic interaction. This was observed when the mother was Specc1l∆CCD2/+. However, remarkably, if the mother was Thm1aln/+, there was no CP incidence in Specc1l∆CCD2/+ and Specc1l∆CCD2/+;Thm1aln/+ embryos even though they had shortened cilia, suggesting Thm1aln/+ mothers provided a protective environment against CP phenotype. Consistent with our previous data, Specc1l∆Ex4;Thm1aln/+ mice resulted in no CP. These results suggest that shortened cilia inSpecc1l mutants do not cause but rather modulate CP phenotype. Overall, our studies indicate a novel role for SPECC1L in ciliogenesis, a novel genetic interaction between Specc1l andThm1, and a novel protective maternal genetic effect of Thm1 in palatogenesis.

Select Amino Acids Increase Anion Secretion in Human Bronchial Epithelial Cells (HBEC) with F508del and Nonsense Mutations: Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis

ObjectiveChloride and bicarbonate transport across the apical membrane via CFTR is critical for homeostasis of airway surface liquid and mucociliary clearance. Cystic fibrosis (CF) is caused by mutations in the CFTR gene resulting in mucus buildup, bacterial colonization, inflammation and respiratory insufficiency. F508del and nonsense mutations are the most common mutations characterized by defective protein processing and trafficking (F508del) or early termination of CFTR synthesis (G542X, R1162X). A combination of CFTR modulators (VX445/661/770; VX) has largely improved CFTR function in patients with F508del but is ineffective in nonsense mutations. We have shown that select amino acids (SAA) can modulate trafficking and activity of CFTR and alternative anion‐secretory pathways, and thus could improve clinical outcomes in CF patients.HypothesisSAA can stimulate anion secretion in primary HBEC with F508del/F508del, F508del/R1162X and G542X/G542X mutation.MethodsTransepithelial short‐circuit current (Isc) was measured in differentiated HBEC treated with DMSO or VX for 24h and bathed in vehicle or SAA in Ussing chambers. After blocking ENaC with benzamil, benzamil‐insensitive (anion) Isc was either continuously recorded for 90min, or sequentially blocked with CFTRinh172, CaCCinhA01 and bumetanide in 15min intervals. Isotope (36Cl) flux studies and whole‐cell patch clamp technique were used to confirm chloride movements, and CFTR and alternative anion‐secretory channel expression was analyzed by immunofluorescence.ResultsAnion Isc increased in all mutations and was more sustained when exposed to SAA compared to vehicle (F508del: 15.4±0.5 vs 1.4±0.2; R1162X: 19.4±0.3 vs 2.3±0.3; G542X: 17.2±0.5 vs 1.6±0.2 µA.cm‐2; P<0.001). SAA recovered 48.7, 57.3 and 40.9% of normal Isc (21.9±0.5 µA.cm‐2). Treatment with VX increased anion Isc in F508del and R1162X (11.4±0.7 and 5.7±0.4 µA.cm‐2; P<0.001) but not in G542X (1.7±0.2 µA.cm‐2). In F508del, the CFTR response increased from 20.7% (non‐treated cells) to 32.9 and 85.5% of the anion Isc when treated with SAA or VX, and in R542X from 3.7% to 46.3 and 16.0%, respectively. Both, SAA and VX did not affect CaCC, but SAA increased the bumetanide‐sensitive Isc from 31.0 to 50.5% in F508del, and from 33.3 to 56.3% in R1162X, while VX reduced the bumetanide response to 5.4 and 4.5%. Isotope flux studies showed that SAA (‐0.21±0.08 μEq.h‐1.cm‐2) and VX (‐0.19±0.02 μEq.h‐1.cm‐2) increased net Cl‐ secretion in F508del when compared to vehicle (‐0.03±0.01 μEq.h‐1.cm‐2; P<0.05). SAA, but not VX increased Cl‐ secretion in G542X (‐0.21 0.07 vs ‐0.08±0.02 μEq.h‐1.cm‐2). In patch clamp recordings, SAA and VX resulted in a significant reduction in CFTRinh172 whole cell current in F508del. Immunofluorescence imaging demonstrated increased CFTR and alternative anion‐secretory channel expression along the apical membrane in SAA cells while VX showed increased expression of only CFTR.ConclusionsThese studies demonstrate that SAA stimulates anion current and increases Cl‐ secretion via CFTR and alternative secretory pathways in different mutations, and could be beneficial in nonsense mutations but needs further investigation.

Activation of the unfolded protein response by Connexin47 mutations associated with Pelizaeus-Merzbacher-like disease

Pelizaeus-Merzbacher-like disease type 1 (PMLD1) is a hypomyelinating disorder arising in patients with mutations in GJC2, encoding Connexin47 (Cx47). PMLD1 causes nystagmus, cerebellar ataxia, spasticity and changes in CNS white matter detected by MRI. At least one mutation (p.I33M) yields a much milder phenotype, spastic paraplegia type 44 (SPG44). Cx47 contributes to gap junction communication channels between oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), and between OLs and astrocytes. Prior studies in cell lines have shown that PMLD1 mutants such as p.P87S display defective protein trafficking, intracellular retention in the ER and loss-of-function. Here we show that when expressed in primary OLs, three PMLD1 associated mutants (p.P87S, p.Y269D and p.M283T) show ER retention of Cx47 and evidence of activation of the cellular stress (unfolded protein response, UPR) and apoptotic pathways. On the other hand, the milder SPG44 associated mutation p.I33M shows a wild-type-like subcellular distribution and no activation of the UPR or apoptotic pathways. These studies provide new insight into a potential element of toxic gain of function underlying the mechanism of PMLD1 that should help guide future therapeutic approaches.

Alcohol-Induced Liver Injury: Down-regulation and Redistribution of Rab3D Results in Atypical Protein Trafficking.

Previous work from our laboratories has identified multiple defects in endocytosis, protein trafficking, and secretion, along with altered Golgi function after alcohol administration. Manifestation of alcohol‐associated liver disease (ALD) is associated with an aberrant function of several hepatic proteins, including asialoglycoprotein receptor (ASGP‐R), their atypical distribution at the plasma membrane (PM), and secretion of their abnormally glycosylated forms into the bloodstream, but trafficking mechanism is unknown. Here we report that a small GTPase, Rab3D, known to be involved in exocytosis, secretion, and vesicle trafficking, shows ethanol (EtOH)–impaired function, which plays an important role in Golgi disorganization. We used multiple approaches and cellular/animal models of ALD, along with Rab3D knockout (KO) mice and human tissue from patients with ALD. We found that Rab3D resides primarily in trans‐ and cis‐faces of Golgi; however, EtOH treatment results in Rab3D redistribution from trans‐Golgi to cis‐medial‐Golgi. Cells lacking Rab3D demonstrate enlargement of Golgi, especially its distal compartments. We identified that Rab3D is required for coat protein I (COPI) vesiculation in Golgi, and conversely, COPI is critical for intra‐Golgi distribution of Rab3D. Rab3D/COPI association was altered not only in the liver of patients with ALD but also in the donors consuming alcohol without steatosis. In Rab3D KO mice, hepatocytes experience endoplasmic reticulum (ER) stress, and EtOH administration activates apoptosis. Notably, in these cells, ASGP‐R, despite incomplete glycosylation, can still reach cell surface through ER‐PM junctions. This mimics the effects seen with EtOH‐induced liver injury. Conclusion : We revealed that down‐regulation of Rab3D contributes significantly to EtOH‐induced Golgi disorganization, and abnormally glycosylated ASGP‐R is excreted through ER‐PM connections, bypassing canonical (ER→Golgi→PM) anterograde transportation. This suggests that ER‐PM sites may be a therapeutic target for ALD.

Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

REP1 deficiency causes systemic dysfunction of lipid metabolism and oxidative stress in choroideremia.

Choroideremia (CHM) is an X-linked recessive chorioretinal dystrophy caused by mutations in CHM, encoding for Rab escort protein 1 (REP1). Loss of functional REP1 leads to the accumulation of unprenylated Rab proteins and defective intracellular protein trafficking, the putative cause for photoreceptor, retinal pigment epithelium (RPE), and choroidal degeneration. CHM is ubiquitously expressed, but adequate prenylation is considered to be achieved, outside the retina, through the isoform REP2. Recently, the possibility of systemic features in CHM has been debated; therefore, in this study, whole metabolomic analysis of plasma samples from 25 CHM patients versus age- and sex-matched controls was performed. Results showed plasma alterations in oxidative stress–related metabolites, coupled with alterations in tryptophan metabolism, leading to significantly raised serotonin levels. Lipid metabolism was disrupted with decreased branched fatty acids and acylcarnitines, suggestive of dysfunctional lipid oxidation, as well as imbalances of several sphingolipids and glycerophospholipids. Targeted lipidomics of the chmru848 zebrafish provided further evidence for dysfunction, with the use of fenofibrate over simvastatin circumventing the prenylation pathway to improve the lipid profile and increase survival. This study provides strong evidence for systemic manifestations of CHM and proposes potentially novel pathomechanisms and targets for therapeutic consideration.

Hepatocyte proteomes reveal the role of protein disulfide isomerase 4 in alpha 1-antitrypsin deficiency

Background & AimsA single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention.MethodsOur functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease.ResultsWe report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease.ConclusionsPDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD.Lay summaryProtein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.

Regulation and biological function of metal ions in Drosophila.

A conceptual framework is offered for critically approaching the formidable ability of insects to segregate metal ions to their multiple destinations in proteins and subcellular compartments. New research in Drosophila melanogaster suggests that nuclear iron regulatory proteins and oxidative stress transcription factors mediate metal-responsive gene expression. Identification of a zinc-regulated chaperone in the endoplasmic reticulum potentially explains membrane protein trafficking defects observed in zinc transporter mutants. Compartmentalized zinc is utilized in fertilization, embryogenesis and for the activation of zinc-finger transcription factors - the latter function demonstrated during muscle development, while dietary zinc is sensed through gating of a chloride channel. Another emerging theme in cellular metal homeostasis is that transporters and related proteins meet at endoplasmic reticulum-mitochondria associated membranes with physiologically relevant consequences during aging.

Understanding protein trafficking defects

Alzheimer's Disease Alzheimer's disease (AD) is characterized by abnormal protein accumulation in the extracellular milieu in the brain. The endosomal pathway is responsible for proteolytic cleavage and secretion of proteins, and recent data have shown that a defective endosomal pathway contributes to AD pathogenesis. However, which proteins are affected by defective endosomal pathways is still unclear. Simoes et al. performed proteomic analysis in cerebrospinal fluid from mice with defective endosomal trafficking and in patients with AD and showed that the expression of two transmembrane proteins, APLP1 and CHL, was increased and correlated with tau expression. The results provide valuable insights regarding the effect of endosomal trafficking alterations in AD. Sci. Transl. Med. 12 , eaba6334 (2020).

Usher Syndrome: Genetics and Molecular Links of Hearing Loss and Directions for Therapy.

Usher syndrome (USH) is an autosomal recessive (AR) disorder that permanently and severely affects the senses of hearing, vision, and balance. Three clinically distinct types of USH have been identified, decreasing in severity from Type 1 to 3, with symptoms of sensorineural hearing loss (SNHL), retinitis pigmentosa (RP), and vestibular dysfunction. There are currently nine confirmed and two suspected USH-causative genes, and a further three candidate loci have been mapped. The proteins encoded by these genes form complexes that play critical roles in the development and maintenance of cellular structures within the inner ear and retina, which have minimal capacity for repair or regeneration. In the cochlea, stereocilia are located on the apical surface of inner ear hair cells (HC) and are responsible for transducing mechanical stimuli from sound pressure waves into chemical signals. These signals are then detected by the auditory nerve fibers, transmitted to the brain and interpreted as sound. Disease-causing mutations in USH genes can destabilize the tip links that bind the stereocilia to each other, and cause defects in protein trafficking and stereocilia bundle morphology, thereby inhibiting mechanosensory transduction. This review summarizes the current knowledge on Usher syndrome with a particular emphasis on mutations in USH genes, USH protein structures, and functional analyses in animal models. Currently, there is no cure for USH. However, the genetic therapies that are rapidly developing will benefit from this compilation of detailed genetic information to identify the most effective strategies for restoring functional USH proteins.