My favorite orthologs

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Human TNF

This human TNF gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine.

For a recent review of the human TNF superfamily see Aggarwal et al, 2012.

TNF superfamily members include the cytokines: TNF (TNF-alpha), LT (lymphotoxin-alpha, TNF-beta), CD40 ligand, Apo2L (TRAIL), Fas ligand, and osteoprotegerin (OPG) ligand. These proteins generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues.

TNF superfamily structure

They initiate apoptosis by binding to related receptors, some of which have intracellular death domains. They generally form homo- or hetero- trimeric complexes.TNF cytokines bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer with ligand trimerization a requiste for receptor binding.

Orthologs

TNF is highly conserved among mammals, with over 90% homology in primates. It is highly homologous to the murine (mus musculus TNF, accession number NP_038721.1), showing a 79% homology.

An initial search for orthologs did not bring many hits. I kept combing the literature and found a reference to a molecule called Eiger in Drosophila and another reference to a TNF-like molecule in the sea urchin. Then I kept mining and found references to TNF in fishes and amphibians.

<I have to make a philosophical pause here. Scientists are usually very specialized in their fields, a necessary requirement. Specialization comes often with complete blindness to other disciplines or areas. So I confess that during all my years of researching TNF I did not look further than the “TNF is highly conserved in vertebrates” mantra, so focused I was on human TNF and human diseases associated to TNF. It was only when writing my thesis that I ventured into reading more in detail about other organisms, as many mechanistical details of TNF pathways came from comparing immune pathways in different organisms, particularly in Drosophila. So I am glad to say that although I chose TNF as an example of this assignment because I thought it would be slam-dunk to prepare, I have actually learned a lot of cool information that was new for me.>

Finally I have arrived to this article by Wiens and Glenney, 2011, which I just ordered through ScienceDirect, so I do not have access to it…but here is what the abstract says:

The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in gene number primarily through multiple whole genome duplication events as well as by lineage and/or species-specific tandem duplication and translocation. The identification and functional analyses of teleost ligands and receptors provide insight into the critical transition between invertebrates and higher vertebrates. Bioinformatic analyses of fish genomes and EST datasets identify 14 distinct ligand groups, some of which are novel to teleosts, while to date, only limited numbers of receptors have been characterized in fish. The most studied ligand is TNF of which teleost species possess between 1 and 3 copies as well as a receptor similar to TNFR1. Functional studies using zebrafish indicate a conserved role of this ligand–receptor system in the regulation of cell survival and resistance to infectious disease.

I need to show this- a comparison of the vertebrate and the amphibious immune system, copied from Huang’s article. See TNF to the left of both.

Slide showing a comparison between amphibian and vertebrate immune systems

Comparison between amphibian and vertebrate immune systems (from Huang et al, 2008).

Well, I have to stop here…but I hope you see my point of the importance of narrowing down your topic, as it always expands as you start digging!

I hope you undertand a bit the point of the past 2 assignments: we are looking for a protein related to a function. The structure of the protein is often related to its function- and we have had the chance to see different proteins, from transcription factors to toxins, enzymes, and hormones. Take a pause and look at the function- how specific it is? That will relate directly to how conserved that gene is going to be- you would expect a protein involved in some essential and widespread metabolic function to go back earlier in phylogeny compared to a protein associated to a more specific function. In my particular example, TNF is part of the immune response, so it is mainly described for vertebrates particularly mammals. But as you see from the abstract above, you see the beginnings going back to Arthropods.

Conserved features

Trimer interface, which is a conserved ring of hydrophobic residues; aids in self-assembly of monomers.

Receptor binding site. TNF ligands share a common structural motif, the TNF homology domain (THD), which binds to cysteine-rich domains (CRDs) of TNF receptors. CRDs are composed of structural modules, whose variation in number and type confers heterogeneity upon the family. Protein folds reminiscent of the THD and CRD are also found in other protein families, raising the possibility that the mode of interaction between TNF and TNF receptors might be conserved in other contexts

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My favorite protein assignment: tumor necrosis factor (TNF)

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This is an example of how to organize the protein assignment page.

Human Tumor necrosis factor 

Tumor necrosis factor (also called cachectin, TNF-alpha, or TNFSF2) is a cytokine originally described as a mediator of septic shock but it is currently considered a master regulator of cell death, survival, and organogenesis.

Tumor necrosis factor (TNF; also named TNFa) is a type II transmembrane protein with an intracellular amino terminus. It has signalling potential both as a 26 kd membrane-integrated protein and as a soluble cytokine released after cleavage by the protease TACE; its soluble form is a trimer of 17 kDa components. There are two TNF receptors: TNFR1, which is found on most cells in the body, and TNFR2, which is primarily expressed on cells of hematopoietic origin. TNFR1 is activated by both TNF forms, while TNFR2 primarily binds transmembrane TNF. TNF receptors are also shed and act as soluble TNF-binding proteins, competing with cell surface receptors for free ligand and thus inhibiting TNF action (Locksley et al, 2001, Hehlgans et al, 2005).

The signaling pathways mediated by the two receptors are slightly divergent. TNFR1 is considered to  mediate more systemic effects. The result of its activation can lead to cell proliferation or death depending on context.  In contrast to TNFR1, TNFR2 lacks a death domain.  Its biological role  is still not fully understood, although recent evidence suggests that it can modulate the actions of TNFR1 on immune and endothelial cells. Transmembrane TNF can function as both ligand and receptor: soluble TNF receptors can bind to the cytokine on the cell surface and generate reverse signaling (Balkwill, 2009).

figure of TNF amino acid sequence

TNF Sequence (from PDB)

Gene

The human TNF gene (TNFA) was cloned in 1985 (Lloyd et al, 1985). It maps to chromosome 6p21.3 (short arm),  close or within the MHC (Major Histocompatibility Complex) region. It spans about 3 kb and contains 4 exons. The 3′ UTR of TNF alpha contains an AU-rich element (ARE), providing a means of post-transcriptional control.

Protein

The protein is translated as a 233 amino acid (26kD) type II transmembrane protein, which is further cleaved by the protease TACE (ADAM 17). Both forms exist as trimers. The 17 kd TNF protomers (185-amino acid-long) are composed of two antiparallel β-pleated sheets with antiparallel β-strands, forming a ‘jelly roll’ β-structure, typical for the TNF family.

Here are two renderings of TNF from the PDB site. However, for the 3D effects you may want to visit the Jmol view.

PDB rendering of TNF

PDB rendering of TNF

molecular view of TNF

Another rendering of TNF

Protein Databank Reference (PDB): 1TNF 

Uniprot entry: P01375 

NCBI RefSeq : NP000585.2

My favorite protein assignment: tumor necrosis factor (TNF)

Leave a comment

This is an example of how to organize the protein assignment page.

Human Tumor necrosis factor 

Tumor necrosis factor (also called cachectin, TNF-alpha, or TNFSF2) is a cytokine originally described as a mediator of septic shock but it is currently considered a master regulator of cell death, survival, and organogenesis.

Tumor necrosis factor (TNF; also named TNFa) is a type II transmembrane protein with an intracellular amino terminus. It has signalling potential both as a 26 kd membrane-integrated protein and as a soluble cytokine released after cleavage by the protease TACE; its soluble form is a trimer of 17 kDa components. There are two TNF receptors: TNFR1, which is found on most cells in the body, and TNFR2, which is primarily expressed on cells of hematopoietic origin. TNFR1 is activated by both TNF forms, while TNFR2 primarily binds transmembrane TNF. TNF receptors are also shed and act as soluble TNF-binding proteins, competing with cell surface receptors for free ligand and thus inhibiting TNF action (Locksley et al, 2001, Hehlgans et al, 2005).

The signaling pathways mediated by the two receptors are slightly divergent. TNFR1 is considered to  mediate more systemic effects. The result of its activation can lead to cell proliferation or death depending on context.  In contrast to TNFR1, TNFR2 lacks a death domain.  Its biological role  is still not fully understood, although recent evidence suggests that it can modulate the actions of TNFR1 on immune and endothelial cells. Transmembrane TNF can function as both ligand and receptor: soluble TNF receptors can bind to the cytokine on the cell surface and generate reverse signaling (Balkwill, 2009).

figure of TNF amino acid sequence

TNF Sequence (from PDB)

Gene

The human TNF gene (TNFA) was cloned in 1985 (Lloyd et al, 1985). It maps to chromosome 6p21.3 (short arm),  close or within the MHC (Major Histocompatibility Complex) region. It spans about 3 kb and contains 4 exons. The 3′ UTR of TNF alpha contains an AU-rich element (ARE), providing a means of post-transcriptional control.

Protein

The protein is translated as a 233 amino acid (26kD) type II transmembrane protein, which is further cleaved by the protease TACE (ADAM 17). Both forms exist as trimers. The 17 kd TNF protomers (185-amino acid-long) are composed of two antiparallel β-pleated sheets with antiparallel β-strands, forming a ‘jelly roll’ β-structure, typical for the TNF family.

Here are two renderings of TNF from the PDB site. However, for the 3D effects you may want to visit the Jmol view.

PDB rendering of TNF

PDB rendering of TNF

molecular view of TNF

Another rendering of TNF

Protein Databank Reference (PDB): 1TNF 

Uniprot entry: P01375 

NCBI RefSeq : NP000585.2

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