Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.     

The Leukocyte Nuclear Envelope Proteome Varies with Cell Activation and Contains Novel Transmembrane Proteins That Affect Genome Architecture

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.The nuclear envelope (NE)1 is a double membrane system consisting of the intermediate filament nuclear lamin polymer and associated proteins attached to the inner nuclear membrane (INM) (1), nuclear pore complexes (NPCs) that direct transport of soluble macromolecules in and out of the nucleus (2), and the outer nuclear membrane (ONM) and associated proteins. Structurally, the ONM is continuous with the endoplasmic reticulum (ER) and is studded with ribosomes (3), yet it also contains unique proteins, many of which connect the cytoskeleton to the NE (4). On the other side, lamins and many INM proteins directly connect chromatin to the NE. Lamins and an increasing number of nuclear envelope transmembrane proteins (NETs) have been linked to a similarly increasing number of diseases ranging from muscular dystrophy to neuropathy, dermopathy, lipodystrophy, bone disorders, and progeroid aging syndromes (5, 6).A favored hypothesis to explain how different NE proteins can produce such a wide range of disease pathologies is that chromatin-NE connections are disrupted with NE protein mutations, yielding changes in gene regulation. This hypothesis is supported by observations that the distribution of dense peripheral chromatin is affected in fibroblasts from patients with NE-linked muscular dystrophy, cardiomyopathy, mandibuloacral dysplasia, and progeria (7–10). Furthermore, many binding partners have been identified for NETs that are either chromatin proteins, enzymes that modify chromatin proteins, or regulators of gene expression (1, 11). These include markers of silent chromatin such as heterochromatin protein 1 (12) and proteins that modify chromatin to a silent conformation such as histone deacetylase 3 (13). The importance of the NE to global genome organization has been underscored by several recent studies that showed that affinity-based recruitment of a specific chromosome locus by the NE both pulled entire chromosomes to the periphery and affected gene regulation in complex ways (14–16).To identify NE proteins likely to be involved in genome organization, we turned to lymphocytes as a model system. Lymphocytes in the resting state tend to have massive amounts of dense peripheral chromatin as determined by electron microscopy studies. Upon activation with phytohemagglutinin, this dense chromatin largely dissipates as the cells actively express genes (17–20). Thus, to identify proteins that might be involved in tethering heterochromatin to the NE or in changing its organization, we analyzed the NE proteomes of leukocyte populations (∼70% lymphocytes) in both the resting and phytohemagglutinin (PHA)-activated states. The previously validated subtractive approach was applied (21) using microsomes and mitochondria, the principal membrane contaminants expected, as subtractive fractions.Many new NE proteins were identified that had not been identified in previous NE proteomics investigations using liver and neuroblastoma cells (21, 22). NE residence was confirmed for 12 novel NETs by expression of epitope-tagged versions and using antibodies on tissue cryosections.Roughly one-quarter of the proteins identified varied between the resting and activated states. Some NET differences between the two data sets were confirmed by RT-PCR. Among the known proteins identified were several that suggest that changes in NE composition associated with PHA activation contribute to gene regulation. Novel NETs identified also appear to play significant roles in genome organization/regulation as we found that several can either recruit a specific locus to the nuclear periphery or promote chromatin condensation. As several studies have implicated misregulation of chromatin organization in NE diseases (7, 8), these newly identified NETs may contribute to the diverse pathologies associated with NE diseases.     

Differential Expression and Function of Stamp Family Proteins in Adipocyte Differentiation

Six transmembrane protein of prostate (Stamp) proteins play an important role in prostate cancer cell growth. Recently, we found that Stamp2 has a critical role in the integration of inflammatory and metabolic signals in adipose tissue where it is highly expressed and regulated by nutritional and metabolic cues. In this study, we show that all Stamp family members are differentially regulated during adipogenesis: whereas Stamp1 expression is significantly decreased upon differentiation, Stamp2 expression is increased. In contrast, Stamp3 expression is modestly changed in adipocytes compared to preadipocytes, and has a biphasic expression pattern during the course of differentiation. Suppression of Stamp1 or Stamp2 expression both led to inhibition of 3T3-L1 differentiation in concert with diminished expression of the key regulators of adipogenesis - CCAAT/enhancer binding protein alpha (C/ebpα) and peroxisome proliferator-activated receptor gamma (Pparγ). Upon Stamp1 knockdown, mitotic clonal expansion was also inhibited. In contrast, Stamp2 knockdown did not affect mitotic clonal expansion, but resulted in a marked decrease in superoxide production that is known to affect adipogenesis. These results suggest that Stamp1 and Stamp2 play critical roles in adipogenesis, but through different mechanisms.     

Interactions among Drosophila Nuclear Envelope Proteins Lamin, Otefin, and YA

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH₂-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.     

Cytoplasmic Capes Are Nuclear Envelope Intrusions That Are Enriched in Endosomal Proteins and Depend upon βH-Spectrin and Annexin B9

It is increasingly recognized that non-erythroid spectrins have roles remote from the plasma membrane, notably in endomembrane trafficking. The large spectrin isoform, β_H, partners with Annexin B9 to modulate endosomal processing of internalized proteins. This modulation is focused on the early endosome through multivesicular body steps of endocytic processing and loss of either protein appears to cause a traffic jam before removal of ubiquitin at the multivesicular body. We previously reported that β_H/Annexin B9 influenced EGF receptor signaling. While investigating this effect we noticed that mSptiz, the membrane bound precursor of the secreted EGF receptor ligand sSpitz, is located in striking intrusions of the nuclear membrane. Here we characterize these structures and identify them as ‘cytoplasmic capes’, which were previously identified in old ultrastructural studies and probably coincide with recently recognized sites of non-nuclear-pore RNA export. We show that cytoplasmic capes contain multiple endosomal markers and that their existence is dependent upon β_H and Annexin B9. Diminution of these structures does not lead to a change in mSpitz processing. These results extend the endosomal influence of β_H and its partner Annexin B9 to this unusual compartment at the nuclear envelope.     

The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.The hepatitis B virus (HBV) is the main human pathogen responsible for severe hepatic diseases like cirrhosis and hepatocellular carcinoma. Even though infection can be prevented by immunization with an efficient vaccine, about 2 billion people have been infected worldwide, resulting in 350 million chronic carriers that are prone to develop liver diseases (56). Current treatments consist either of the use of interferon α, which modulates antiviral defenses and controls infection in 30 to 40% of cases, or of the use of viral polymerase inhibitors that allow a stronger response to treatment but require long-term utilization and frequently lead to the outcome of resistant viruses (34, 55). A better understanding of the virus life cycle, and particularly of the mechanism by which the virus enters the cell, could provide background for therapeutics that inhibit the early steps of infection, as recently illustrated with the HBV pre-S1-derived entry inhibitor (25, 45).HBV belongs to the Hepadnaviridae family whose members infect different species. All viruses of this family share common properties. The capsid containing a partially double-stranded circular DNA genome is surrounded by a lipid envelope, in which two (in avihepadnaviruses infecting birds) or three (in orthohepadnaviruses infecting mammals) envelope proteins are embedded. A single open reading frame bearing several translation initiation sites encodes these surface proteins. Thus, the HBV envelope contains three proteins: S, M, and L that share the same C-terminal extremity corresponding to the small S protein that is crucial for virus assembly (7, 8, 46) and infectivity (1, 31, 53). These proteins are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles through the Golgi apparatus (15, 42). The current model for the transmembrane structure of the S domain implies the luminal exposition of both N- and C-terminal extremities and the presence of four transmembrane (TM) domains: the TM1 and TM2 domains, both necessary for cotranslational protein integration into the ER membrane, and the TM3 and TM4 domains, located in the C-terminal third of the S domain (for a review, see reference 6). Among the four predicted TM domains, only the TM2 domain has a defined position between amino acids 80 and 98 of the S domain. The exact localization of the TM1 domain is still unclear, probably because of the relatively low hydrophobicity of its sequence, which contains polar residues and two prolines. The M protein corresponds to the S protein extended by an N-terminal domain of 55 amino acids called pre-S2. Its presence is dispensable for both assembly and infectivity (20, 21, 37). Finally, the L protein corresponds to the M protein extended by an N-terminal domain of 108 amino acids called pre-S1 (genotype D). The pre-S1 and pre-S2 domains of the L protein can be present either at the inner face of viral particles (on the cytoplasmic side of the ER), playing a crucial role in virus assembly (5, 8, 10, 11, 46), or on the outer face (on the luminal side of the ER), available for the interaction with target cells and necessary for viral infectivity (4, 14, 36). The pre-S translocation is independent from the M and S proteins and is driven by the L protein TM2 domain (33). Finally, HBV surface proteins are not only incorporated into virion envelopes but also spontaneously bud from ER-Golgi intermediate compartment membranes (30, 43) to form empty subviral particles (SVPs) that are released from the cell by secretion (8, 40).One approach to decipher viral entry is to interfere with the function of envelope proteins. Thus, by a mutagenesis approach, two envelope protein domains crucial for HBV infectivity have already been identified: (i) the 77 first amino acids of the pre-S1 domain (4, 36) including the myristic acid at the N-terminal extremity (9, 27) and (ii) possibly a cysteine motif in the luminal loop of the S domain (1, 31). In addition, a putative fusion peptide has been identified at the N-terminal extremity of the S domain due to its sequence homology with other viral fusion peptides (50). This sequence, either N-terminal in the S protein or internal in the L and M proteins, is conserved among the Hepadnaviridae family and shares common structural and functional properties with other fusion peptides (49, 50). Finally, a PEST sequence likely containing several proteolytic cleavage sites has been identified in the L and M proteins upstream of the TM1 domain (39). A cleavage within this sequence could activate the fusion peptide.In this study, we investigated whether the putative fusion peptide and the PEST sequence were necessary for the infection process. For this purpose, we constructed a set of mutant viruses bearing contiguous deletions in these regions and determined their infectivity using an in vitro infection model based on HepaRG cells (28). The introduction of mutations either in the L and M proteins or in only the S protein allowed us to demonstrate that, in the TM1 domain of L protein, three hydrophobic clusters not essential for viral assembly were crucial for HBV infectivity while their presence in the S protein was critical for envelope protein expression. In addition, we showed that the PEST sequence was clearly dispensable for both assembly and infection processes.     

The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.     

A Simple Method for Predicting Transmembrane Proteins Based on Wavelet Transform

The increasing protein sequences from the genome project require theoretical methods to predict transmembrane helical segments (TMHs). So far, several prediction methods have been reported, but there are some deficiencies in prediction accuracy and adaptability in these methods. In this paper, a method based on discrete wavelet transform (DWT) has been developed to predict the number and location of TMHs in membrane proteins. PDB coded as 1KQG is chosen as an example to describe the prediction process by this method. 80 proteins with known 3D structure from Mptopo database are chosen at random as data sets (including 325 TMHs) and 80 sequences are divided into 13 groups according to their function and type. TMHs prediction is carried out for each group of membrane protein sequences and obtain satisfactory result. To verify the feasibility of this method, 80 membrane protein sequences are treated as test sets, 308 TMHs can be predicted and the prediction accuracy is 96.3%. Compared with the main prediction results of seven popular prediction methods, the obtained results indicate that the proposed method in this paper has higher prediction accuracy.     

TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia

Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals under age 65. In many patients, the predominant pathology includes neuronal cytoplasmic or intranuclear inclusions of ubiquitinated TAR DNA binding protein 43 (FTLD‐TDP). Recently, a genome‐wide association study identified the first FTLD‐TDP genetic risk factor, in which variants in and around the TMEM106B gene (top SNP rs1990622) were significantly associated with FTLD‐TDP risk. Intriguingly, the most significant association was in FTLD‐TDP patients carrying progranulin (GRN) mutations. Here, we investigated to what extent the coding variant, rs3173615 (p.T185S) in linkage disequilibrium with rs1990622, affects progranulin protein (PGRN) biology and transmembrane protein 106 B (TMEM106B) regulation. First, we confirmed the association of TMEM106B variants with FTLD‐TDP in a new cohort of GRN mutation carriers. We next generated and characterized a TMEM106B‐specific antibody for investigation of this protein. Enzyme‐linked immunoassay analysis of progranulin protein levels showed similar effects upon T185 and S185 TMEM106B over‐expression. However, over‐expression of T185 consistently led to higher TMEM106B protein levels than S185. Cycloheximide treatment experiments revealed that S185 degrades faster than T185 TMEM106B, potentially due to differences in N‐glycosylation at residue N183. Together, our results provide a potential mechanism by which TMEM106B variants lead to differences in FTLD‐TDP risk.

     

A Novel Assay for Assessing Juxtamembrane and Transmembrane Domain Interactions Important for Receptor Heterodimerization

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin α_IIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.     

细胞水平筛选鉴定激活核因子κB通路的新基因TMEM9B

核因子κB(NF-κB)是细胞内重要的转录因子,其介导的细胞信号转导通路在细胞凋亡中的作用是国内外研究的热点．为了筛选NF-κB通路相关新基因,建立了基于细胞水平的报告基因高通量筛选模型．利用双荧光素酶报告系统检测报告基因荧光素酶活性,通过对构建的439个人类未知功能基因的筛选,获得了一批激活NF-κB信号通路的功能基因,其中基因TMEM9B可以明显激活NF-κB通路．进一步实验显示TMEM9B激活NF-κB通路呈明显剂量依赖性,Western blot及EMSA实验证实,TMEM9B能够促进胞质内NF-κB的抑制分子IκBα的降解,并促使NF-κB由胞质向胞核转移,同时流式细胞术实验发现TMEM9B可引起293T和HeLa细胞的凋亡．总之,所建立的基于细胞水平的NF-κB通路筛选模型稳定高效,筛选并验证TMEM9B可明显激活NF-κB信号转导通路,并从而引起细胞凋亡．     

Formation of Sindbis Virus Proteins: Identification of a Precursor for One of the Envelope Proteins

Exposure of Sindbis virus-infected chicken embryo cells to a short pulse of radioactive amino acids revealed the formation of primarily three proteins: the nucleocapsid (C) of the virus, one of the viral envelope proteins (E1), and a glycoprotein that did not appear in the virion. This third protein (PE2) has now been identified as a precursor of the other viral envelope protein (E2) on the basis of two observations: (i) the simultaneous disappearance of radioactive PE2 and appearance of labeled E2 in pulse-chase experiments, and (ii) the identity of (14)C-arginine tryptic peptides in fingerprints of the two proteins. The nucleocapsid was the most heavily labeled protein in the cell and appeared in the virus during the short pulse. The two (14)C-labeled envelope proteins, although having different kinetics of labeling in the cell, appeared simultaneously in the virus only after the chase. Addition of pactamycin, a drug inhibiting initiation of protein synthesis, preferentially inhibited the formation of capsid protein Assuming that Sindbis virus proteins are formed initially as a single polypeptide, our studies locate the nucleocapsid at the amino-terminal end of the polypeptide chain.