Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin.

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs act as actin-activated NC toxins. Here, we explore the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) instead of filamentous (F-actin) actin.

Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date.

Many pathogens manipulate host cell cAMP signaling pathways to promote their survival and proliferation. Bacterial Exoenzyme Y (ExoY) toxins belong to a family of invasive, structurally-related bacterial nucleotidyl cyclases (NC). Inactive in bacteria, they use proteins that are uniquely and abundantly present in eukaryotic cells to become potent, unregulated NC enzymes in host cells.

How HIV-1 Integrase Associates with Human Mitochondrial Lysyl-tRNA Synthetase.

Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNA from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNA, an essential step to initiate reverse transcription in the virions. The C-terminal integrase moiety of GagPol is essential for its association with mLysRS.

Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl-tRNA synthetase activity.

Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme.

Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA.

Background: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.

Association of human mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol does not require other viral proteins.

In human, the cytoplasmic (cLysRS) and mitochondrial (mLysRS) species of lysyl-tRNA synthetase are encoded by a single gene. Following HIV-1 infection, mLysRS is selectively taken up into viral particles along with the three tRNA isoacceptors. The GagPol polyprotein precursor is involved in this process.

Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor.

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle.

Association of mitochondrial Lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of Pol.

Cytosolic and mitochondrial lysyl-tRNA synthetases (LysRS) are encoded by a single gene and can be distinguished only according to their very N-terminal sequences. It was believed that cytosolic LysRS is packaged into HIV-1 virions via its association with Gag. Using monospecific antibodies, it was later shown that only the mitochondrial LysRS is taken up in viral particles along with tRNA(3)(Lys), the primer for reverse transcription of the HIV-1 genome.

Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform.

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector.

Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin.

Purpose: To characterize the activities of irofulven, a novelanticancer agent derived from the mushroom natural productilludin S toward human cancer cells.

Experimental Design: We have determined the activity spectrum of irofulven toward a human tumor cell panel comprised of 10 different tumor types in comparison with cisplatin and ET-743. We have also evaluated the influence of major resistance mechanisms, such as expression of multidrug resistance-associated drug efflux pumps, cisplatin resistance, loss of p53 function, and absence of mismatch repair on the cytotoxic activity of irofulven.