-SLENP: A Chemical Genetics Strategy To Selectively Label Existing Proteins and Newly Synthesized Proteins.

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as -SLENP.

-SLENP: A chemical genetics strategy to selectively label existing proteins and newly synthesized proteins.

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as -SLENP.

Nutrient sensing in Leishmania: Flagellum and cytosol.

Parasites are by definition organisms that utilize resources from a host to support their existence, thus, promoting their ability to establish long-term infections and disease. Hence, sensing and acquiring nutrients for which the parasite and host compete is central to the parasitic mode of existence. Leishmania are flagellated kinetoplastid parasites that parasitize phagocytic cells, principally macrophages, of vertebrate hosts and the alimentary tract of sand fly vectors.

A cytoskeletal protein complex is essential for division of intracellular amastigotes of .

Previous studies in have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON.

Touching the Surface: Diverse Roles for the Flagellar Membrane in Kinetoplastid Parasites.

While flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a multitude of functions at this interface: attachment of parasites to tissues within insect vectors, close interactions with intracellular organelles of vertebrate cells, transactions between flagella from different parasites, junctions between the flagella and the parasite cell body, emergence of nanotubes and exosomes from the parasite directed to either host or microbial targets, immune evasion, and sensing of the extracellular milieu.

MAF1b Binds the Host Cell MIB Complex To Mediate Mitochondrial Association.

Many diverse intracellular pathogens, such as , , sp., and , manipulate and relocate host cell organelles, including mitochondria. tachyzoites use a secreted protein, mitochondrial association factor 1b (MAF1b), to drive the association between the host mitochondria and the membrane of the parasitophorous vacuole, in which the parasites grow.

A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.

Unlabelled: The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction.

Impact of regulated secretion on antiparasitic CD8 T cell responses.

CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole.

Quantitative control of protein S-palmitoylation regulates meiotic entry in fission yeast.

Protein S-palmitoylation, a lipid modification mediated by members of the palmitoyltransferase family, serves as an important membrane-targeting mechanism in eukaryotes. Although changes in palmitoyltransferase expression are associated with various physiological and disease states, how these changes affect global protein palmitoylation and cellular function remains unknown. Using a bioorthogonal chemical reporter and labeling strategy to identify and analyze multiple cognate substrates of a single Erf2 palmitoyltransferase, we demonstrate that control of Erf2 activity levels underlies the differential modification of key substrates such as the Rho3 GTPase in vegetative and meiotic cells.

De novo growth zone formation from fission yeast spheroplasts.

Eukaryotic cells often form polarized growth zones in response to internal or external cues. To understand the establishment of growth zones with specific dimensions we used fission yeast, which grows as a rod-shaped cell of near-constant width from growth zones located at the cell tips. Removing the cell wall creates a round spheroplast with a disorganized cytoskeleton and depolarized growth proteins.

Spatial control of Cdc42 activation determines cell width in fission yeast.

The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it an ideal system for studying the control of cellular dimensions. In this study, we carried out a near-genome-wide screen for mutants wider than wild-type cells.

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites.

Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.

The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration.