Journal: PLoS ONE

Article Title: Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

doi: 10.1371/journal.pone.0016156

Figure Lengend Snippet: (A) Exogenous expression of Wild type LdUfm1 and the mutant forms. Leishmania transfectants overexpressing either wild type full length Ufm1 (Ufm1 WT ) or truncated form in which 18 aminoacid residues at the C-terminus including Gly 98 were removed (Ufm1 ΔC ) or the mutant form in which the putative cleavage site Gly 98 is altered (Ufm1 G98A ) were prepared. The transfectants Ufm1 WT , Ufm1 ΔC and Ufm1 G98A carried an HA epitope tag at the N-terminus. Protein lysates from equal number of the transfectants (Uba5 WT , Uba5 C217S and Uba5 C217A ) and L. donovani wild type parasites (WT) were resolved on SDS-PAGE and probed with anti-HA antibodies (α-HA). (B) IgG fraction enriched from the polyclonal antibodies raised against L. donovani Uba5 protein were reacted with promastigote L. donovani lysates (α-LdUba5) in an immunoblot. Pre-immune serum was used as a control (NRS). (C) Leishmania transfectants overexpressing either wild type Uba5 (Uba5 WT ) or the mutant forms (Uba5 C217S , Uba5 C217A ) in which the predicted active site Cys was changed to either Ser or Ala by PCR were prepared. The transfectants Uba5 WT and Uba5 C217A possessed an HA epitope tag at the C-terminus while in the Uba5 C217S mutant, the HA tag was at the N-terminus. Protein lysates from equal number of L. donovani wild type parasites (WT) and the transfectants (Uba5 WT , Uba5 C217S and Uba5 C217A ) were resolved on SDS-PAGE and probed with either with polyclonal antibodies raised against LdUba5 (α-LdUba5) or with anti-HA antibodies (α-HA). (D) Identification of the stable intermediate linked to LdUba5 in Leishmania transfectant cells. Leishmania transfectants overexpressing Ufm1 containing both 6xHis and HA tags at the N-terminus (Ufm1 WT ) or co-expressing Ufm1 WT and either wild type Uba5 (Uba5 WT ) or the mutant forms (Uba5 C217S , Uba5 C217A ) were prepared. Protein lysates from equal number of L. donovani transfectants expressing Ufm1 WT alone or co-expressing variants of Uba5 (Uba5 WT , Uba5 C217S and Uba5 C217A ) were resolved on SDS-PAGE and probed with either with polyclonal antibodies raised against LdUba5 (α-LdUba5) or with anti-His antibodies (α-His). (E) Immunoblot analysis following immunoprecipitation reactions. Protein lysates from wild type L. donovani cells (WT) or transfectants expressing variants of LdUfm1 (Ufm1 WT , Ufm1 G98A or Ufm1 ΔC ) were used in co-immunoprecipitation reactions with α-HA affinity gel and the eluates were resolved on SDS-PAGE and the immunoblots were probed with anti-LdUba5 antibodies (α-LdUba5). Ponceau-S stained immunoblot showing the heavy chain IgG (IgG H/C) to indicate the amount of antibodies used in immunoprecipitation reactions. The immunoblot of the total lysates with α-LdUba5 antibodies indicating equal amounts of lysates used in immunoprecipitation reaction is shown. (F) Conversely, protein lysates from wild type L. donovani cells (WT) or transfectants (Ufm1 WT , Ufm1 G98A or Ufm1 ΔC ) were used in co-immunoprecipitation reactions with α-LdUBA5 antibodies and the immunoblots were probed with anti-HA antibodies (α-HA). Ponceau-S stained immunoblot showing the heavy chain IgG (IgG H/C) to indicate the amount of antibodies used in immunoprecipitation reactions.

Article Snippet: Human recombinant Ufm1 and Uba5 were purchased from Boston Biochem, Cambridge, MA.

Techniques: Expressing, Mutagenesis, SDS Page, Western Blot, Control, Transfection, Immunoprecipitation, Staining

Journal: PLoS ONE

Article Title: Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

doi: 10.1371/journal.pone.0016156

Figure Lengend Snippet: (A) Activation of Leishmania Ufm1 by LdUba5. Leishmania Ufm1 lacking the 17 aminoacid residues at the C-terminus and thus exposing the terminal Gly residue and a mutant Ufm1 in which the terminal Gly is changed to Ala were immunoprecipitated from the respective Leishmania transfectants were incubated with either wild type LdUba5 or LdUba5 C217A proteins immunoprecipitated from the corresponding Leishmania transfectants in the combinations indicated either with or without ATP and changes in the magnitude of fluorescent polarization are measured. Leishmania proteins from LdUba5, LdUba5 C217A , LdUfm1 or LdUfm1 G98A transfectants were included as background controls. The data are collected from three independent experiments. Error bars indicate the standard deviation. (B) Activation of human Ufm1 by LdUba5. Human Ufm1 was incubated with either wild type LdUba5 or LdUba5 C217A proteins immunoprecipitated from the corresponding Leishmania transfectants and ATP and changes in the magnitude of fluorescent polarization are measured. Leishmania proteins from LdUba5 or LdUba5 C217A transfectants without human Ufm1 were included as background controls. (C) Human recombinant Uba5 and Ufm1 proteins were incubated either in the presence or absence of ATP and changes in the magnitude of fluorescent polarization in the tracer molecule are measured indicating the activity of Uba5.

Article Snippet: Human recombinant Ufm1 and Uba5 were purchased from Boston Biochem, Cambridge, MA.

Techniques: Activation Assay, Residue, Mutagenesis, Immunoprecipitation, Incubation, Standard Deviation, Recombinant, Activity Assay

Journal: PLoS ONE

Article Title: Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

doi: 10.1371/journal.pone.0016156

Figure Lengend Snippet: (A) Human macrophages differentiated from monocytes were infected with purified metacyclic parasites from L. donovani wild type (WT) or transfectant cultures (Ufm1, Ufm1 G98A or Ufm1 ΔC ) for six hours (10∶1 parasite-to-macrophage ratio) and the numbers of amastigotes in these cultures were determined over a period of 6 days by microscopic observation of Diff-quik reagent stained slides. The data are expressed as the number of amastigotes per 100 macrophages. Error bars indicate the standard deviation. (B) The growth of L. donovani wild-type (WT), or wild-type Uba5 (LdUba5) or mutant Uba5 transfectants (LdUba5 C217S or LdUba5 C217A ) amastigotes in human macrophages was monitored. The results are the mean of three independent experiments.

Article Snippet: Human recombinant Ufm1 and Uba5 were purchased from Boston Biochem, Cambridge, MA.

Techniques: Infection, Purification, Transfection, Diff-Quik, Staining, Standard Deviation, Mutagenesis

Journal: PLoS ONE

Article Title: Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

doi: 10.1371/journal.pone.0016156

Figure Lengend Snippet: (A) Affinity purified polyclonal antibodies raised against T. brucei Ufm1 protein reacted with promastigote L. donovani lysates (α-Ufm1) in an immunoblot. Only a shorter exposure of the immunoblot is shown to avoid revealing the multiple faint bands resulting from Ufm1 conjugates. Pre-immune serum was used as a control (NRS). (B) L. donovani promastigote cells were stained with antibodies against TbUfm1 (α-Ufm1) as primary, and Alexa488-conjugated anti-rabbit IgG as secondary antibodies. To visualize mitochondria, cells were labeled with Mitotracker Red (Mito Red). The nucleus and kinetoplast were stained with DAPI. Pre-immune serum (NRS) was used in control staining experiments. (C) L. donovani promastigote cells were stained with IgG fraction enriched from anti-LdUba5 antiserum (α-LdUba5) as primary, and Alexa488-conjugated anti-rabbit IgG as secondary antibodies. Mitochondria, nucleus and kinetoplast were stained as described above. Pre-immune serum (NRS) was used in control staining experiments. (D) Affinity purified polyclonal antibodies raised against L. donovani Ufc1 protein were reacted with promastigote L. donovani lysates (α-LdUfc1) in an immunoblot. Pre-immune serum was used as a control (NRS). (E) L. donovani promastigote cells were stained with antibodies against LdUfc1 (α-LdUfc1) as primary, and Alexa488-conjugated anti-rabbit IgG as secondary antibodies. Mitochondria, nucleus and kinetoplast were stained as described above. Pre-immune serum (NRS) was used in control staining experiments. Bars, 5 µm (B, C) 10 µm (E).

Article Snippet: Human recombinant Ufm1 and Uba5 were purchased from Boston Biochem, Cambridge, MA.

Techniques: Affinity Purification, Western Blot, Control, Staining, Labeling

Journal: PLoS ONE

Article Title: Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

doi: 10.1371/journal.pone.0016156

Figure Lengend Snippet: (A) Protein lysates either from promastigote stage (Pro) or axenic amastigote (AxAm) stage Leishmania transfectant cells expressing either HA-His-Ufm1 WT or HA-His-Ufm ΔC were prepared under denaturing conditions and the conjugates were precipitated with Ni-agarose beads. The eluates from the beads were subjected to SDS-PAGE and the immunoblots were probed with α-Ufm1 antibodies. Pre-immune serum (NRS) was used in control immunoblots. (B) Leishmania transfectant cells expressing HA-His-Ufm1 WT or the non-conjugatable HA-His-Ufm ΔC were lysed under denaturing conditions and the conjugates were precipitated with Ni-agarose beads. The eluates from the beads were subjected to SDS-PAGE stained with Coomassie blue and scanned on a LiCor odyssey instrument. The SDS-gel slices excised from the acrylamide gel for the purpose of peptide identification are indicated with white arrow marks. (C) The identification of the peptides from the gel slices and the corresponding proteins to which the peptides map to are listed. The asterisks (B and C) indicate the gel slices from which the peptides originated.

Article Snippet: Human recombinant Ufm1 and Uba5 were purchased from Boston Biochem, Cambridge, MA.

Techniques: Transfection, Expressing, SDS Page, Western Blot, Control, Staining, SDS-Gel, Acrylamide Gel Assay