Targeting Fn14 as a therapeutic target for cachexia reprograms the glycolytic pathway in tumour and brain in mice.

Purpose: Cachexia is a complex syndrome characterized by unintentional weight loss, progressive muscle wasting and loss of appetite. Anti-Fn14 antibody (mAb 002) targets the TWEAK receptor (Fn14) in murine models of cancer cachexia and can extend the lifespan of mice by restoring the body weight of mice. Here, we investigated glucose metabolic changes in murine models of cachexia via [F]FDG PET imaging, to explore whether Fn14 plays a role in the metabolic changes that occur during cancer cachexia.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs.

Fn14: a new player in cancer-induced cachexia.

Purpose Of Review: Although cancer cachexia is a very significant condition that is present in up to 80% of cancer cases, the cause of the condition has remained a puzzle. Cancer cachexia is a condition which is mainly characterised by muscle wasting, mobilization of fat reserves, and overall metabolic disturbance. This review aims to highlight some of the recent findings in cancer cachexia research.

New method for purifying histidine-rich glycoprotein from human plasma redefines its functional properties.

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography.

Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import.

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex.

Discovery of genes activated by the mitochondrial unfolded protein response (mtUPR) and cognate promoter elements.

In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebp beta gene is similarly activated and CHOP and C/EBP beta subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes.

The chop gene contains an element for the positive regulation of the mitochondrial unfolded protein response.

We have previously reported on the discovery of a mitochondrial specific unfolded protein response (mtUPR) in mammalian cells, in which the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional activation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ, and ClpP, but not those encoding stress proteins of the endoplasmic reticulum (ER) or the cytosol. Analysis of the chaperonin 60/10 bidirectional promoter showed that the CHOP element was required for the mtUPR and that the transcription of the chop gene is activated by mtUPR. In order to investigate the role of CHOP in the mtUPR, we carried out a deletion analysis of the chop promoter.

Mitochondrial-nuclear communications.

Mitochondria cannot be made de novo but replicate by a mechanism of recruitment of new proteins, which are added to preexisting subcompartments. Although mitochondria have their own DNA, more than 98% of the total protein complement of the organelle is encoded by the nuclear genome. Mitochondrial biogenesis requires a coordination of expression of two genomes and therefore cross talk between the nucleus and mitochondria.

Mitochondrial morphology and distribution in mammalian cells.

It is now appreciated that mitochondria form tubular networks that adapt to the requirements of the cell by undergoing changes in their shape through fission and fusion. Proper mitochondrial distribution also appears to be required for ATP delivery and calcium regulation, and, in some cases, for cell development. While we now realise the great importance of mitochondria for the cell, we are only beginning to work out how these organelles undergo the drastic morphological changes that are essential for cellular function.

Characterisation of the protein composition of peripheral blood mononuclear cell microsomes by SDS-PAGE and mass spectrometry.

Approximately 340 leucocyte plasma membrane proteins have been characterised by the eight Human Leucocyte Differentiation Antigen workshops held between 1982 and 2004, based primarily on their reactivity with monoclonal antibodies. The human genome is predicted to encode approximately 34,000 cDNA transcripts, of which between 15% and 20% are predicted to contain one or more transmembrane helices. We have used SDS-PAGE separation coupled with mass spectrometry-based peptide mass tag identification to identify novel plasma membrane proteins in microsome preparations prepared from mononuclear cells obtained from human peripheral blood.

Dissection of the mitochondrial import and assembly pathway for human Tom40.

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane.

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection.

Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70.

The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase.

Import of nuclear-encoded proteins into mitochondria.

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes. In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components. We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment.

A mitochondrial specific stress response in mammalian cells.

Cells respond to a wide variety of stresses through the transcriptional activation of genes that harbour stress elements within their promoters. While many of these elements are shared by genes encoding proteins representative of all subcellular compartments, cells can also respond to stresses that are specific to individual organelles, such as the endoplasmic reticulum un folded protein response. Here we report on the discovery and characterization of a mitochondrial stress response in mammalian cells.

Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane.

Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space.

Import and assembly of proteins into mitochondria of mammalian cells.

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing fungal systems. This review outlines recent advances in our understanding of this process in mammalian cells. In particular, we focus on the characterisation of cytosolic molecular chaperones that are involved in binding to mitochondrial-targeted preproteins, as well as the identification of both conserved and novel subunits of the import machineries of the outer and inner mitochondrial membranes.