Zika Virus Capsid Anchor Forms Cytotoxic Amyloid-like Fibrils

Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During ZIKV polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Since misfolding and aggregation propensity of transmembrane proteins are now increasingly recognized and has been linked to several proteopathic disorders. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed aggregation behavior of CA peptide using dyebinding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM and AFM) revealed characteristic amyloid-like fibrils. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection.Graphical Abstract:Schematic representation of Zika virus Capsid anchor forming amyloid aggregates with cytotoxic and hemolytic properties.

Protein Translocation Acquires Substrate Selectivity Through ER Stress-Induced Reassembly of Translocon Auxiliary Components

Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

ABSTRACT Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC. To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres. We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

Characterization of Intermediate Steps in Amyloid Beta (Aβ) Production under Near-native Conditions

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aβ peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aβ and in particular Aβ42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aβ production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aβ40 and Aβ42 and an increase in shorter Aβ peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aβ51 to Aβ31/Aβ30 and from Aβ49 to Aβ30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aβ48 to Aβ38 compared with Aβ49 to Aβ37. These findings correlate with an increase in the Aβ42/40 ratio. GSMs caused a decrease in Aβ40 and Aβ42 and an increase in Aβ37 and Aβ38 paralleled by an increase of the intermediates Aβ40–38 and Aβ42–39. Collectively, these data provide a thorough characterization of all intermediate steps in Aβ production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aβ generation.

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.