The recombinant L-lysine α-oxidase from the fungus Trichoderma harzianum promotes apoptosis and necrosis of leukemia CD34 + hematopoietic cells.

BACKGROUND: In hematologic cancers, including leukemia, cells depend on amino acids for rapid growth. Anti-metabolites that prevent their synthesis or promote their degradation are considered potential cancer treatment agents. Amino acid deprivation triggers proliferation inhibition, autophagy, and programmed cell death. L-lysine, an essential amino acid, is required for tumor growth and has been investigated for its potential as a target for cancer treatment. L-lysine α-oxidase, a flavoenzyme that degrades L-lysine, has been studied for its ability to induce apoptosis and prevent cancer cell proliferation. In this study, we describe the use of L-lysine α-oxidase (LO) from the filamentous fungus Trichoderma harzianum for cancer treatment. RESULTS: The study identified and characterized a novel LO from T. harzianum and demonstrated that the recombinant protein (rLO) has potent and selective cytotoxic effects on leukemic cells by triggering the apoptotic cascade through mitochondrial dysfunction. CONCLUSIONS: The results support future translational studies using the recombinant LO as a potential drug for the treatment of leukemia.

Expression, purification, and characterization of the TRIM49 protein.

Autophagy is the process of degradation of intracellular proteins through the lysosome. Members of the tripartite motif (TRIM) proteins have shown to directly recognize autophagic cargo and also to act as a hub for the phagophore nucleation complex. The TRIM proteins are classically characterized by the presence of an amino-terminal RING domain and a B-box domain followed by a coiled coil domain. Although regarded as ubiquitin E3 ligases, this activity has been shown only for a minor set of the 79 human TRIM proteins. Additionally, the role of each domain in the E3 ligase activity is unknown. We investigated the role of the SPRY and RING domains of the human TRIM49 protein in its E3 ubiquitin ligase activity. Wild-type and mutant constructs of tagged TRIM49 were expressed in E. coli or mammalian cells, and the autoubiquitination activity of the purified protein was assessed. The purified TRIM49 showed no ubiquitin E3 ligase activity in vitro. However, cells transfected with the wild-type or mutant protein showed increased levels of lower mass polyubiquitinated proteins and both proteins copurified with polyubiquitinated proteins. Taken together, these results indicate that the TRIM49 protein plays a role in autophagic protein degradation independently of an ubiquitin E3 ligase activity.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction.

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XlnR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xlnA and xlnD mRNA accumulation. The ΔfbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi.

Modulation of the Host Nuclear Compartment by Trypanosoma cruzi Uncovers Effects on Host Transcription and Splicing Machinery.

Host manipulation is a common strategy for invading pathogens. Trypanosoma cruzi, the causative agent of Chagas Disease, lives intracellularly within host cells. During infection, parasite-associated modifications occur to the host cell metabolism and morphology. However, little is known about the effect of T. cruzi infection on the host cell nucleus and nuclear functionality. Here, we show that T. cruzi can modulate host transcription and splicing machinery in non-professional phagocytic cells during infection. We found that T. cruzi regulates host RNA polymerase II (RNAPII) in a time-dependent manner, resulting in a drastic decrease in RNAPII activity. Furthermore, host cell ribonucleoproteins associated with mRNA transcription (hnRNPA1 and AB2) are downregulated concurrently. We reasoned that T. cruzi may hijack the host U2AF35 auxiliary factor, a key regulator for RNA processing, as a strategy to affect the splicing machinery activities directly. In support of our hypothesis, we carried out in vivo splicing assays using an adenovirus E1A pre-mRNA splicing reporter, showing that intracellular T. cruzi directly modulates the host cells by appropriating U2AF35. For the first time, our results provide evidence of a complex and intimate molecular relationship between T. cruzi and the host cell nucleus during infection.

Identification of FBXO25-interacting proteins using an integrated proteomics approach.

FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.

Ischemic preconditioning of renal tissue: identification of early up-regulated genes.

Given the important effects of ischemic preconditioning (IPC) in minimizing tissue damage induced by sustained ischemia in several tissues, this study evaluated the effect of IPC in preserving renal function and identified up-regulated genes after 30 min of preconditioning. IPC induced by 2, 3 and 4 min of ischemia, intercalated by 5 min of reperfusion, induced a measurable protection of renal function and morphology. The improved functional and histological parameters occurred in parallel with up-regulation of 39 genes, as evaluated by subtractive hybridization; for 13 of them we could show, by RNAse protection assay, a significant increase in mRNA levels. These genes code for chaperones/chaperonins and cytoskeleton proteins that could be involved in preservation of protein folding and cellular structures after sustained ischemia; proteins related to oxidative metabolism that might be relevant for cellular use of alternate sources of energy or for faster recovery of ATP levels in this condition, and proteins that are putative scavengers of oxidant products. Summarizing, ischemic preconditioning induced up-regulation of genes that code proteins whose functional roles suggest their involvement in the tolerance of the preconditioned tissue to sustained ischemia.

Mouse Leydig cells express multiple P2X receptor subunits.

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 muM ivermectin induced an increase (131.2 +/- 5.9%) and 3 muM ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

Ubiquitin-ligase and deubiquitinating gene expression in stretched rat skeletal muscle.

In order to gain insight into intracellular mechanisms involved in longitudinal growth of skeletal muscle, we determined gene expression of ubiquitin-ligases (MAFbx/atrogin-1, E3 alpha, and MuRF-1) and deubiquitinating enzymes (UBP45, UBP69, and USP28) at different time-points (24, 48, and 96 h) of continuous stretch of the soleus and tibialis anterior (TA) muscles. In the soleus, real-time polymerase chain reaction (PCR) showed that MAFbx/atrogin-1, E3 alpha, and MuRF-1 gene expression was downregulated, peaking at 24-48 h. Gene expression of all deubiquitinating enzymes increased with continuous stretch of soleus. In the TA, gene expression of the ubiquitin-ligases MAFbx/atrogin-1 and MuRF-1 was elevated, whereas expression of UBP45 and UBP69 was downregulated. Western blot analysis showed that the overall ubiquitination level decreased in the soleus and increased in the TA during stretch. These results suggest that ubiquitin-ligases and deubiquitinating enzymes are involved in longitudinal growth induced by continuous muscle stretch.