Short transmembrane domains target type II proteins to the Golgi apparatus and type I to the endoplasmic reticulum.

Transmembrane domains (TMDs) contain information targeting membrane proteins to various compartments of the secretory pathway. In previous studies, short or hydrophilic TMDs have been shown to target membrane proteins either to the endoplasmic reticulum (ER), or to the Golgi apparatus. The basis for differential sorting to the ER and to the Golgi apparatus remained however unclear. To clarify this point, we analyzed quantitatively the intracellular targeting of a collection of proteins exhibiting a single TMD. Our results reveal that membrane topology is a major targeting element in the early secretory pathway: type I proteins with a short transmembrane domain are targeted to the ER, and type II proteins to the Golgi apparatus. A combination of three features accounts for the sorting of simple membrane proteins in the secretory pathway: membrane topology, length and hydrophilicity of the TMD, and size of the cytosolic domain. By clarifying the rules governing sorting to the ER and to the Golgi apparatus, our study may revive the search for sorting mechanisms in the early secretory pathway.

The C-terminal sequences of Bcl-2 family proteins mediate interactions that regulate cell death.

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP.

Assignment of the Lassa virus transmembrane domain in the prefusion and postfusion states in detergent micelles.

Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa, and recently isolated cases worldwide, there are no current therapeutics or vaccines. As such, LASV poses a significant global public health threat. One of the key steps in LASV infection is delivering its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Previously, we showed that the TM undergoes pH-dependent structural changes that result in a helical extension. Here, we provide the 1H, 15N, and 13C assignment of the LASV TM backbone in the prefusion and postfusion states. We also provide the 1H, 15N, and 13C assignment of two mutants, G429P and D432P, which prevent this helical extension. These results will help understand the role the TM plays in membrane fusion and can lead to the design of therapeutics against LASV infection.

Identification of amino acids in transmembrane domains of mutated cytokine receptor-like factor 2 and interleukin-7 receptor α required for constitutive signal transduction.

Cytokine receptor-like factor 2 (CRLF2) and interleukin-7 receptor α (IL-7Rα) form a receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation consisting of the substitution of five amino acids (SLLLL) in the transmembrane domain of CRLF2 with three amino acids, including glutamic acid, isoleucine, and methionine (insEIM), which has been identified in acute lymphocytic leukemia, causes the TSLP-independent dimerization with IL-7Rα and activation. However, the dimerization mechanism remains unclear. In this study, we examined the involvement of the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα in TSLP-independent activation. HEK293 cells were transfected with vectors encoding CRLF2 and IL-7Rα, or their mutants, in which the amino acid of the transmembrane domain was replaced with alanine. STAT5 phosphorylation was detected using western blotting, and receptor dimerization was analyzed using the NanoBiT assay. The substitution of glutamic acid within the insEIM mutation for alanine failed to cause the STAT5 phosphorylation in the absence of TSLP. Moreover, the alanine substation of the specific leucine residues in the transmembrane domains of both CRLF2 and IL-7Rα abrogated the TSLP-independent signal transduction and dimerization. The mutation of IL-7Rα W264 partially reduced the phosphorylation of STAT5 without affecting receptor dimerization. These results suggest that the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα play at least three possible functions: interaction through hydrogen bonds, hydrophobic interaction, and signal transduction. Our findings contribute to a better understanding of the function of the transmembrane domains of cytokine receptors in their dimerization and signal transduction.

Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated with oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the stability of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

The topological model of NS4B and its TMD3 in duck TMUV proliferation.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Crystallization of Ethylene Plant Hormone Receptor-Screening for Structure.

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium-high resolution of 2-4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling.

The full-length TSH receptor is stabilized by TSH ligand.

The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the "hinge region", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Research advances of MAL family members in tumorigenesis and tumor progression (Review).

The myelin and lymphocyte protein (MAL) family is a novel gene family first identified and characterized in 2002. This family is comprised of seven members, including MAL, MAL2, plasmolipin, MALL, myeloid differentiation­associated marker (MYADM), MYADML2 and CMTM8, which are located on different chromosomes. In addition to exhibiting extensive activity during transcytosis, the MAL family plays a vital role in the neurological, digestive, respiratory, genitourinary and other physiological systems. Furthermore, the intimate association between MAL and the pathogenesis, progression and metastasis of malignancies, attributable to several mechanisms such as DNA methylation has also been elucidated. In the present review, an overview of the structural and functional properties of the MAL family and the latest research findings regarding the relationship between several MAL members and various cancers is provided. Furthermore, the potential clinical and scientific significance of MAL is discussed and directions for future research are summarized.

Studying Conformational Properties of Transmembrane Domain of KCNE3 in a Lipid Bilayer Membrane Using Molecular Dynamics Simulations.

KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility.

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.

Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimerization.

Receptor protein tyrosine phosphatases (RPTPs) are one of the key regulators of receptor tyrosine kinases (RTKs) and therefore play a critical role in modulating signal transduction. While the structure-function relationship of RTKs has been widely studied, the mechanisms modulating the activity of RPTPs still need to be fully understood. On the other hand, homodimerization has been shown to antagonize RPTP catalytic activity and appears to be a general feature of the entire family. Conversely, their documented ability to physically interact with RTKs is integral to their negative regulation of RTKs, but there is a yet-to-be proposed common model. However, specific transmembrane (TM) domain interactions and residues have been shown to be essential in regulating RPTP homodimerization, interactions with RTK substrates, and activity. Therefore, elucidating the contribution of the TM domains in RPTP regulation can provide significant insights into how these receptors function, interact, and eventually be modulated. This chapter describes the dominant-negative AraC-based transcriptional reporter (DN-AraTM) assay to identify specific TM interactions essential to homodimerization and heteroassociation with other membrane receptors, such as RTKs.

Septins throughout phylogeny are predicted to have a transmembrane domain, which in Caenorhabditis elegans is functionally important.

Septins, a conserved family of filament-forming proteins, contribute to eukaryotic cell division, polarity, and membrane trafficking. Septins are thought to act in these processes by scaffolding other proteins to the plasma membrane. The mechanisms by which septins associate with the plasma membrane are not well understood but can involve two polybasic domains and/or an amphipathic helix. We discovered that the genomes of organisms throughout phylogeny, but not most commonly used model organisms, encode one or more septins predicted to have transmembrane domains. The nematode Caenorhabditis elegans, which was thought to express only two septin proteins, UNC-59 and UNC-61, translates multiple isoforms of UNC-61, and one isoform, UNC-61a, is predicted to contain a transmembrane domain. UNC-61a localizes specifically to the apical membrane of the C. elegans vulva and is important for maintaining vulval morphology. UNC-61a partially compensates for the loss of the other two UNC-61 isoforms, UNC-61b and UNC-61c. The UNC-61a transmembrane domain is sufficient to localize a fluorophore to membranes in mammalian cells, and its deletion from UNC-61a recapitulates the phenotypes of unc-61a null animals. The localization and loss-of-function phenotypes of UNC-61a and its transmembrane domain suggest roles in cell polarity and secretion and help explain the cellular and tissue biological underpinnings of C. elegans septin null alleles' enigmatically hypomorphic phenotypes. Together, our findings reveal a novel mechanism of septin-membrane association with profound implications for the dynamics and regulation of this association.

A Novel Form of Neuregulin 1 Type III Caused by N-Terminal Processing.

Nrg1 (Neuregulin 1) type III, a susceptible gene of schizophrenia, exhibits a critical role in the central nervous system and is essential at each stage of Schwann's cell development. Nrg1 type III comprises double-pass transmembrane domains, with the N-terminal and C-terminal localizing inside the cells. The N-terminal transmembrane helix partially overlaps with the cysteine-rich domain (CRD). In this study, Nrg1 type III constructs with different tags were transformed into cultured cells to verify whether CRD destroyed the transmembrane helix formation. We took advantage of immunofluorescent and immunoprecipitation assays on whole cells and analyzed the N-terminal distribution. Astonishingly, we found that a novel form of Nrg1 type III, about 10% of Nrg1 type III, omitted the N-terminal transmembrane helix, with the N-terminal positioning outside the membrane. The results indicated that the novel single-pass transmembrane status was a minor form of Nrg1 type III caused by N-terminal processing, while the major form was a double-pass transmembrane status.

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct.

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

Masking the transmembrane region of the amyloid ß precursor protein as a safe means to lower amyloid ß production.

Introduction: Reducing brain levels of both soluble and insoluble forms of amyloid beta (Aß) remains the primary goal of most therapies that target Alzheimer's disease (AD). However, no treatment has so far resulted in patient benefit, and clinical trials of the most promising drug candidates have generally failed due to significant adverse effects. This highlights the need for safer and more selective ways to target and modulate Aß biogenesis. Methods: Peptide technology has advanced to allow reliable synthesis, purification, and delivery of once-challenging hydrophobic sequences. This is opening up new routes to target membrane processes associated with disease. Here we deploy a combination of atomic detail molecular dynamics (MD) simulations, living-cell Förster resonance energy transfer (FRET), and in vitro assays to elucidate the atomic-detail dynamics, molecular mechanisms, and cellular activity and selectivity of a membrane-active peptide that targets the Aß precursor protein (APP). Results: We demonstrate that Aß biogenesis can be downregulated selectively using an APP occlusion peptide (APPOP). APPOP inhibits Aß production in a dose-dependent manner, with a mean inhibitory concentration (IC50) of 450 nM toward exogenous APP and 50 nM toward endogenous APP in primary rat cortical neuronal cultures. APPOP does not impact the γ-secretase cleavage of Notch-1, or exhibit toxicity toward cultured primary rat neurons, suggesting that it selectively shields APP from proteolysis. Discussion: Drugs targeting AD need to be given early and for very long periods to prevent the onset of clinical symptoms. This necessitates being able to target Aß production precisely and without affecting the activity of key cellular enzymes such as γ-secretase for other substrates. Peptides offer a powerful way for targeting key pathways precisely, thereby reducing the risk of adverse effects. Here we show that protecting APP from proteolytic processing offers a promising route to safely and specifically lower Aß burden. In particular, we show that the amyloid pathway can be targeted directly and specificically. This reduces the risk of off-target effects and paves the way for a safe prophylactic treatment.

Development of Zika Virus E Variants for Pseudotyping Retroviral Vectors Targeting Glioblastoma Cells.

A fundamental idea for targeting glioblastoma cells is to exploit the neurotropic properties of Zika virus (ZIKV) through its two outer envelope proteins, prM and E. This study aimed to develop envelope glycoproteins for pseudotyping retroviral vectors that can be used for efficient tumor cell infection. Firstly, the retroviral vector pNLlucAM was packaged using wild-type ZIKV E to generate an E-HIVluc pseudotype. E-HIVluc infection rates for tumor cells were higher than those of normal prME pseudotyped particles and the traditionally used vesicular stomatitis virus G (VSV-G) pseudotypes, indicating that protein E alone was sufficient for the formation of infectious pseudotyped particles. Secondly, two envelope chimeras, E41.1 and E41.2, with the E wild-type transmembrane domain replaced by the gp41 transmembrane and cytoplasmic domains, were constructed; pNLlucAM or pNLgfpAM packaged with E41.1 or E41.2 constructs showed infectivity for tumor cells, with the highest rates observed for E41.2. This envelope construct can be used not only as a tool to further develop oncolytic pseudotyped viruses for therapy, but also as a new research tool to study changes in tumor cells after the transfer of genes that might have therapeutic potential.