Caterina Strambio-De-Castillia

TRIM5 is an innate immune sensor for the retrovirus capsid lattice

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13–UEV1A (also known as UBE2N–UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13–UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13–UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

The nuclear pore complex: bridging nuclear transport and gene regulation.

Although the nuclear pore complex (NPC) is best known for its primary function as the key regulator of molecular traffic between the cytoplasm and the nucleus, a growing body of experimental evidence suggests that this structure participates in a considerably broader range of cellular activities on both sides of the nuclear envelope. Indeed, the NPC is emerging as an important regulator of gene expression through its influence on the internal architectural organization of the nucleus and its apparently extensive involvement in coordinating the seamless delivery of genetic information to the cytoplasmic protein synthesis machinery.

Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1-inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti-HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5alpha, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2-/-gammac-/- mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti-HIV-1 gene therapy candidate.

The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly.

The two yeast proteins Mlp1p and Mlp2p (homologues of the vertebrate protein Tpr) are filamentous proteins attached to the nuclear face of nuclear pore complexes. Here we perform a proteomic analysis, which reveals that the two Mlps have strikingly different interacting partners, testifying to their different roles within the cell. We find that Mlp2p binds directly to Spc110p, Spc42p, and Spc29p, which are three core components of the spindle pole body (SPB), the nuclear envelope–associated yeast spindle organizer. We further show that SPB function is compromised in mlp2 mutants. Cells lacking Mlp2p form significantly smaller SPBs, accumulate aberrant SPB component-containing structures inside the nucleus, and have stochastic failures of cell division. In addition, depletion of Mlp2p is synthetically lethal with mutants impaired in SPB assembly. Based on these data, we propose that Mlp2p links the SPB to the peripheral Mlp assembly, and that this linkage is required for efficient incorporation of components into the SPB.

Processing strategies and software solutions for data‐independent acquisition in mass spectrometry

Data‐independent acquisition (DIA) offers several advantages over data‐dependent acquisition (DDA) schemes for characterizing complex protein digests analyzed by LC‐MS/MS. In contrast to the sequential detection, selection, and analysis of individual ions during DDA, DIA systematically parallelizes the fragmentation of all detectable ions within a wide m/z range regardless of intensity, thereby providing broader dynamic range of detected signals, improved reproducibility for identification, better sensitivity, and accuracy for quantification, and, potentially, enhanced proteome coverage. To fully exploit these advantages, composite or multiplexed fragment ion spectra generated by DIA require more elaborate processing algorithms compared to DDA. This review examines different DIA schemes and, in particular, discusses the concepts applied to and related to data processing. Available software implementations for identification and quantification are presented as comprehensively as possible and examples of software usage are cited. Processing workflows, including complete proprietary frameworks or combinations of modules from different open source data processing packages are described and compared in terms of software availability and usability, programming language, operating system support, input/output data formats, as well as the main principles employed in the algorithms used for identification and quantification. This comparative study concludes with further discussion of current limitations and expectable improvements in the short‐ and midterm future.

The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

Mlp1p and Mlp2p form the basket of the yeast nuclear pore complex (NPC) and contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. The Mlps also embed the NPC within an extended interactome, which includes protein complexes involved in mRNP biogenesis, silencing, spindle organization, and protein degradation.

Isolation of nuclear envelope from Saccharomyces cerevisiae

Publisher Summary The yeast Saccharomyces cerevisiae has been one of the systems of choice for the molecular biologist and the geneticist. This organism has become increasingly amenable to biochemical and cell biological techniques. This chapter discusses the isolation of nuclear envelope from Saccharomyces cerevisiae ( S. cerevisiae ) and presents the modifications of the protocols that are utilized routinely in laboratories for the preparation of nuclei and nuclear envelopes (NEs) from various wild-type and mutant S. cerevisiae strains. In particular, highly enriched yeast nuclei and NEs fractions can be prepared from S. cerevisiae strains that have been modified genetically to encode epitope-tagged versions of genes of interest. This method is highly reproducible in laboratories. The chapter concludes with a description of a faster, cruder method for making nuclei that can be used for preparations in which the highest degree of enrichment is not crucial.

Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS.

Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which, in turn, is detrimental for accurate quantification. The nonoutlier fragment ion (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high-priority fragment ions, these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative data set (i.e., the SWATH Gold Standard) indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to that with the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest data set, NOFI properly assigns low-priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80, against 0.76 for the Spectronaut interference detection algorithm.

Cyclosporine blocks incorporation of HIV-1 envelope glycoprotein into virions.

ABSTRACT Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.

The Structure and Composition of the Yeast NPC

The double-membraned nuclear envelope (NE) behaves as a selective barrier that segregates the genome from all cytosolic processes. A highly regulated exchange system between these two compartments is essential for proper cell growth, progression through the cell cycle, accurate responses to developmental and extracellular signals and to maintain the functional integrity of the nucleus. The sole mediators of controlled nucleocytoplasmic transport are the nuclear pore complexes (NPCs), large proteinaceous machineries embedded within specialized circular pores that traverse the NE. In actively growing cells it is estimated that every minute hundreds of proteins and ribonucleoprotein particles (RNPs) traverse each NPC in both directions. The basic mechanisms of nuclear transport appear to be highly conserved across distantly related species (reviewed in Nigg 1997; Mattaj and Englmeier 1998; Gorlich and Kutay 1999; Wente 2000). Although metabolites, water, ions and small macromolecules can freely diffuse through aqueous channels of 10 nm in the NPC, large macromolecular particles with a diameter of up to 30 nm are selectively transported across the NPC via a highly regulated energy-dependent process. Active transport requires specific soluble transport factors that recognize individual substrates both inside and outside the nucleus and mediate their interaction with the stationary phase of the NPC translocation machinery. Specifically, the translocation of transport substrates is known to require the docking of the transport complex to the NPC, the active translocation of the docked complexes across the NPC and the release of the substrate into the target compartment. Various models have been proposed to explain how this docked complex is actively translocated across the 50–60 nm long NPC transporter and then subsequently released into the nucleoplasm, and the matter is still highly controversial (see below). All models agree in attributing a crucial importance to the protein Ran in maintaining vectorial cargo transport and regulating the binding and release steps that take place during translocation. As a member of the Ras superfamily of small GTPases, Ran exists within the cell in a GDP-bound and in a GTP-bound form. The balance between these two forms is regulated by a variety of Ran cofactors that are asymmetrically distributed within the cell. As a consequence cytoplasmic Ran is thought to exist prevalently in the GDP-bound form, while Ran-GTP is thought to predominate in the nucleus. This differential distribution of Ran-GTP versus Ran-GDP would establish directional transport by ensuring that transport complexes are formed in one compartment and disassembled in the other (reviewed in Cole and Hammell 1998; Mattaj and Englmeier 1998; Pemberton et al. 1998; Wozniak et al. 1998; Gorlich and Kutay 1999). Understanding this regulated transport demands an understanding of the detailed three-dimensional map of the NPC and of the interactions and relationships between the soluble and stationary phases of nuclear transport.