In a living animal without cancer, the extracellular matrix is an odd place to find a cathepsin B-like protein.

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In a living animal without cancer, the extracellular matrix is an odd place to find a cathepsin B-like protein.
Wing phenotype of an adult female containing "clones" of cells lacking the gene being studied.

I am a PhD student in developmental and cell biology.  My studies require a working laboratory knowledge of how to manipulate the genomes of fruit flies to study how cells interact during development by altering gene expression and creating mutant proteins.  This includes a self-sustaining understanding of basic genomics and proteomics, molecular biology, cell culture, developmental processes at the cellular and organismal level, and a natural curiosity to spend enough devoted to figuring out a specific problem in developmental biology.

In my case, I am examining what at intuition says "disease condition," when it's heard.  This condition is the presence of a cathepsin B-like protein in the extracellular matrix.  For those of you not familiar with cathepsins (or protein families in general, for that matter), they are a type of protein that cuts other proteins into smaller pieces, or proteases for short.  Cathepsins are normally found in lysosomes, which are acidic vesicles within cells containing an array of proteases involved in recycling other proteins in the cell.  However, in many cancers, cathepsins can be secreted into the extracellular matrix where they can digest away basement membranes, thus promoting tumor invasion.  So cathepsin B or a cathepsin B-like protein in the extracellular matrix is considered a bad thing.

However it was, to my surprise, that there was an extracellular matrix molecule (a protein) that contained a cathepsin B-like domain that is catalytically inactive.  There is no reported evidence of any activity from the few studies conducted on a mouse version of this protein.  Supporting evidence includes the presence of an amino acid mutation from a cysteine in the active site to a serine (cathepsins belong to a larger family called cysteine proteases).  While there is also a giant family of serine proteases, they do not share sequence homology with cathepsin B.  In experiments with mouse kidneys where this gene was blocked, the epithelial tubules failed to form with much of the epithelial tissue never even differentiating from the mesenchyme.

 So it appears that the structural properties of an inert cathepsin B have become important in the development of multicellular organisms by serving some function outside of cells.  In fruit flies, I see this gene expressed in cells undergoing unique morphogenetic processes during tissue migrations in development.  Through nifty genetic tactics, I have engineered a strain of flies that lack the gene in this extracellular cathepsin b-like protein.  We know it's essentially required in the mother during oogenesis to proceed normally both in terms of egg development and embyro development.  The maternally provided protein to the embryo allows it to be sustained relatively late in larval life when they start to form cysts of their own tissues encapsulated by melanizing "blood cells" in something akin to an immune response.  Removal of the maternally provided RNA through more nifty genetic tactics shows the protein is essential for normal embryonic development (with a small percentage of "escapers" <1%).    

 So the gene appears to be required for development of flies...and of kidneys in more complex vertebrates.  At the cellular level, the protein is possibly serving a very similar purpose.  My aim is to find a specific interaction of this protein with cells and use phenotypic rescue to test which parts of the protein are most vital for proper function.  

I post this as an example of the numerous genes which are conserved not only from flies to humans.  But keep in mind there are genes conserved among bacteria and humans including those responsible for synthesizing ATP, the mitochondrial Fo/F1 ATPase, for the cell's energy requirements.  It's just a small example of changes in genes (duplications and splicing events) that can encode for new proteins with unique functions from pre-existing genes encoding proteins with completely different functions and localization signals.  I can't give away too much information at this point, but we'll be publishing something within in the next year if all the planned experiments, some currently under investigation, support our hypothesis that a particular cell adhesion protein interacts with this protein in these essential morphogenetic events.  Given our understanding of how genetics works, it is not unreasonable to make an inference that some organisms had speciated because of an ability to produce new "mosaic" proteins made of parts of other proteins serving different functions..

I am often amazed at how much more capability and enthusiasm for science there is among elementary school youngsters than among college students.

~Carl Sagan


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Wow. Let me guess.

Wow.

Let me guess. Drosophilia, right? I'm very impressed with those little things, and have lost count of how many successful knockout mutant variants we have of them. Especially the one with an extra set of wings. The genetic homology between humans and Drosophila are quite remarkable, so remarkable that we use it as a model for vertebrae, when it isn't a vertebrae itself.

Actually, when I was first learning about proteomic domains, I was very suprised to learn that there is almost nothing original in the vertebrae genome. It is the result of multiple whole-global duplications throughout evolution. Even in humans, the proteome contains only 7% vertebrae-specific proteins. The only place we really seem to have any originality is in domain shuffling (Im pretty sure that the human tyrpsin can bind to at least 18 domains, while in drosophilia it's only 5)

This is cool research man, and odd. I would never guess that any cathepsins would be found in such a strange place. How far along is it? Have you finished sequencing the molecule you found? How clear is the signature sequence?

"Physical reality” isn’t some arbitrary demarcation. It is defined in terms of what we can systematically investigate, directly or not, by means of our senses. It is preposterous to assert that the process of systematic scientific reasoning arbitrarily excludes “non-physical explanations” because the very notion of “non-physical explanation” is contradictory.

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I am near the end of my 2nd

I am near the end of my 2nd year and am just getting into the meat of things.  My gene is completely sequenced due to the genome project of Drosophila and other organisms (nearly 200 known genome sequences to date).    I'm not sure what you mean by 'signature sequence" unless you mean signal sequence to target proteins for secretion (in which case, it is a classic example).  I think the fly with 2 sets of wings was a null for the ultrabithorax locus, which suppresses certain genes in the posterior set of "halteres" which are really just wing remnants.  Interestingly, I recently heard a talk by a new professor at Clemon University on his work with the Ubx complex.  He is identifying genes targeted by this protein to regulate their transcription in wings fs halteres.  He did introduce the topic by showing the differences between flies, butterflies, and dragonflies with respect to their wings.  Seeing it all in big picture format helps put the individual pieces in place as it is not a challenge to see how the  different genera of insects vary by genetic control. And yes, regarding vertebrates and gene duplication....  Among my favorite molecules are the integrins, which mediate cell attachment to extracellular matrix.  Interestingly, the human has 18 alpha chains and 8 beta chains, all of which are descendent from a common ancestor with flies (as flies have only 5 alpha chains and 2 beta chains).   Relatively chromosomal position of these duplications tell all but when these things occurred. 

I am often amazed at how much more capability and enthusiasm for science there is among elementary school youngsters than among college students.

~Carl Sagan


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I wasnt referring to signal

I wasnt referring to signal sequences. I was referring to signature sequences, as in small stretches of highly conserved amino acids maybe 10-30 long that help ID two protein domains that share a homology, even if it has diverged alot. I asked because I was not sure (it wasnt clear when I read it) whether you had actually discovered a new protein (which wouldn't be very suprising) or found one that's already in the database. If you found a new one, I was interested in the degree of homology which it shared with it's fellow family members by the similiariy found in the domain.

"Physical reality” isn’t some arbitrary demarcation. It is defined in terms of what we can systematically investigate, directly or not, by means of our senses. It is preposterous to assert that the process of systematic scientific reasoning arbitrarily excludes “non-physical explanations” because the very notion of “non-physical explanation” is contradictory.

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No, it has known orthologs

No, it has known orthologs in mammals.  There is the cathepsin b-like domain that I mentioned, but it also has some cysteine motifs similar to those found in extracellular proteins such as follistatin and von willebrand factor.

I am often amazed at how much more capability and enthusiasm for science there is among elementary school youngsters than among college students.

~Carl Sagan


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Honestly, for me this topic

Honestly, for me this topic may as well be written in Russian. Can someone provide a Roseta-stone?


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Well, in the context of

Well, in the context of evolution he is talking about something known as homology. Across the spectrum of life we see very little originality. From the time of the primordial genome, it has expanded in size due to duplication error. This provides the mutation carrier with superfluous genetic baggage, basically an extra copy of a gene. This copy is free to mutate based solely on random frequency probability. It diverges from its original function guided by Natural selection.

This new gene or section will be closely related to the original, both in function and sequence (although divergence starts widening over time). These two genes are said to be paralogous of each other in the same carrier. For instance, the human genome contains seven haemoglobin proteins, all of which are in a gene family called the haemoglobin family. This is part of a larger family called the globin family, under which all oxygen binding proteins are classed like myoglobins.

When two species diverge, the new genetic arm of the phylogenic tree retains much of the genetic code of it's predecessor. Any related batches of genes in two species are said to be orthologous of each other. The seven human haemoglobins are orthologous to the seven chimp ones.

Basically this is how all of evolution works. Genes duplicate by accident, then these new copies diverge in function over time, species branch off, and whole families of related genes spring up. However, when we trace it back to the proto-cells, all the genes are related. There is no such thing as a truly original gene.

This is basically what he is pointing out. The fruit fly Drosophilia is often used as a model of vertebrae organisms, which is odd because it is not a vertebrae. Nonetheless, it matches the vertebrae genome so closely that it can be used for most purposes. Much cheaper to breed than a vertebrae anyway.

The entire vertebrae genome, in fact, is probably the result of a full duplication of an ancestral genome. Some geneticists suggested that it was duplicated twice, producing a quadruple homology.

So what he is explaining is something rather unsuprising, that we have an orthology between a human and a fruit fly in a conserved protein domain.

The other thing he is talking about is bizarre, the prescene of a cathepsin-B in the extracellular matrix, which is the criss-cross of fibrous proteins that keep the integrity of the Eukaryotic lipid bilayer and hold the adhesion junctions of multicellular organisms. That protein is not supposed to be there, unless the animal has cancer.

Well, there is your rosseta stone.

"Physical reality” isn’t some arbitrary demarcation. It is defined in terms of what we can systematically investigate, directly or not, by means of our senses. It is preposterous to assert that the process of systematic scientific reasoning arbitrarily excludes “non-physical explanations” because the very notion of “non-physical explanation” is contradictory.

-Me

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Great, from Russian to

Great, from Russian to Greek.

I think I sorta understand now, but from now on I'll leave the biology to you guys. Undecided


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Great, from Russian to

Great, from Russian to Greek.

Well, yeah. That's what the stone does. 

"Physical reality” isn’t some arbitrary demarcation. It is defined in terms of what we can systematically investigate, directly or not, by means of our senses. It is preposterous to assert that the process of systematic scientific reasoning arbitrarily excludes “non-physical explanations” because the very notion of “non-physical explanation” is contradictory.

-Me

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HAHA!  Beautiful!   Just

HAHA!  Beautiful!

 

Just think of it like today's music artists.  Nothing they do is all that unique.  They're simply either copying what has already been done or putting a small twist on it.

 In the case of biology, you have simple organism like a sponge that has certain genes.  During the life of sponges, their cells divide and new sponges form.  Give them a very very very long time, and copying mistakes will occur during DNA copying when making new sponges.  If those copying mistakes don't kill the new sponge, they can pass them on.  Now suppose a region of one of the sponges chromosomes gets duplicated and inserts itself into the existing chromosomes when otherwise it would be destroyed.  You've just added DNA.  Now suppose it is free to mutate just like the original.

 

Now think back to music.  Take Elvis.  Elvis copied all the black soul artists before him and of his youth.  Whilte people caught on and duplicated Elvis but then started to modify the sound.  The place where Elvis got his ideas...the black community...was changing as well.  Today...we have hip hop and heavy metal.  Something like that...although probably not the best example, but it kinda sorta gets the point across. 

I am often amazed at how much more capability and enthusiasm for science there is among elementary school youngsters than among college students.

~Carl Sagan


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Hey HTR, I am loving what

Hey HTR, I am loving what you are working on - the way you describe it, is the way I've always visualized it. Do keep us posted, and when you are published or ready to publish, let me know, I'd love to take a half hour or so to discuss what you're working on and an hour or so on top of that discussing what it means and why such research is vital.

I am against religion because it teaches us to be satisfied with not understanding the world. - Richard Dawkins

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And I know it's a lot to ask

And I know it's a lot to ask (maybe ask your grad student once you get tenured), but I would love to see a comparison between Stepsipteras, halteras and dipteras.

I am against religion because it teaches us to be satisfied with not understanding the world. - Richard Dawkins

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Yellow_Number_Five

Yellow_Number_Five wrote:
And I know it's a lot to ask (maybe ask your grad student once you get tenured), but I would love to see a comparison between Stepsipteras, halteras and dipteras.

 

HA!  Well, right now I am the graduate student (see the first sentence of the thread!!).  

Anyway, I'm not really an entomologist, though what you're suggesting is indeed interesting.  I would recommend that you look up the work of Sean Carroll (recently invited to join the National Academy of Sciences and is a Howard Hughes Medical Institute investigator at UMW) and keep an eye out for one of his former postdocs/new asst profs at Clemon U, Bradley Hersh  (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17174297&query_hl=2&itool=pubmed_...)..for example.

 

My work is more related to understand how a particular gene functions in development with the idea that it may provide insight into tissue morphogenesis in mammals and potentially provide something toward tissue engineering, in the far reaching implications. 

I am often amazed at how much more capability and enthusiasm for science there is among elementary school youngsters than among college students.

~Carl Sagan


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Thanks for the link, I will

Thanks for the link, I will request and devour said article at my earliest convienience. This sort of research just gets my blood pumping, it's sexy, for lack of a better term. It's the sort of thing that sometimes makes me wish I stuck with biology rather than get into engineering, until I remind myself of the patience this sort of thing requires - patience has never been a virtue of mine.

 Still, I'm intrigued by your work, do keep us posted. I wish you well in your research, and may your centifuge never shatter a vital sample along the way - I know from experience how much that can suck Eye-wink

I am against religion because it teaches us to be satisfied with not understanding the world. - Richard Dawkins

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Hey  man good stuff

Hey  man good stuff here....  some antimalarial drugs or at least potential therapies are thought to interact and disrupt cathespin B and L-like cysteine proteases in the trophozoite stage of the parasite.   During the asexual stage, the parasite is an obligate intraerythrocytic parasite and it has evolved many mechanisms to gain nutrients.  The way it gains free amino acids for protein synthesis is through the digestion of haemoglobin.  Cathepsin B and L like proteases have been found to potentially be responsible for the digestion of haembglobin.  A study has shown that by inhibiting these proteases, the parasite will not grow. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lproteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L.  So whatever this protein(s) is/are (I don't believe it/they has/have been characterized) it/they has/have a highl level of homolgy to cathepsin B and L.  Showing conservation, at least, in part of protein domains.

 Since we're on topic, I'll talk a bit about my research (for a master's thesis).  I'm looking at a putative invasion protein of Plasmodium falciparum that is thought to be an erythrocyte ligand. This protein belongs to a family of proteins called DBL-EBL's (Duffy Binding Like Erythrocyte Binding Ligands)  all these proteins share homology in Region II, which is a cystein rich domain.  I'm trying to characterize this protein.  Without going into too much detail...what I want to underline here is the evolutionary implications for having multiple invasion proteins.  Unlike Plasmodium vivax, which uses only one invasion pathway (via the Duffy Blood Group Antigen), Plasmodium falciparum has multiple invasion pathways.  The interesting aspect of this is the fact that malarial parasites, act as selective pressures on humans. 

In africa, where P. vivax is common, there are plenty of people who are Duffy blood group antigen negative, therefore are refractile to P. vivax invasion, in other words, they're immune, but not in the normal meaning of the word.  P. vivax just cannot invade the erythrocytes of these individuals because the protein that is used in invasion of the red blood cells, the Duffy Binding Ligand (DBL), cannot bind to any other surface protein of the red blood cell therefore will not invade Duffy negative individuals.  This shows that the parasite was acting as a selective pressure on the Duffy blood group gene and because of the high level of death among children in africa with malaria, those that had a mutation that rendered them duffy negative survived and passed that gene along.  AT present only african americans are shown to be duffy blood gropu negative.

However, Plasmodium falciparum, has many pathways for erythrocyte invasion, therefore, bypassing the innate immunity of Duffy negative individuals.  P. falciparum lacks the DBL invasion protein but has homologous proteins which share the erythrocyte binding domain of DBL (Region II).  These proteins bind to different blood group antigens (Sialic acids--specifically Glycophorin A and B and others such as Band 3).  P. falciparum is much more virulent because of it's redundant and multiple invasion pathways of erythrocytes.  There are no known humans that are refractile to P. falciparum.  That is why this class of proteins is called the Duffy binding like-erythrocyte binding ligands, they share homology to the DBL protein but have different binding sites on the red blood cell.

This presents evidence of selection and how the  host (us) also applies selective pressures on these parasites to evolve various invasion pathways.  But it is plain to see that even though these are two separate species, the homology of invasion proteins specific to this phylum is there.  These parasites also share homology with other species such as Cryptosporidium and Trypanosomes.  All of which are under the apicomplexan group (a highly specilized gropu of parasitic protists).

For the benefit of the rest of the members here.  The interesting thing  is the underlying fact that the same family of proteins are found among many taxa of organisms.  Especially if you look at cellular structural proteins.  These are conserved among just about every single living thing on earth.  For example, actin...a cytoskeletal protein is one of the most genetically conserved proteins (by conserved: we mean that there is little change on the genetic make up of this protein throught out all species of life).  The protein actin differs by no more than 5% in all eukaryotes.  

Evolution works slowly but surely as we can see not just with fossil evidence but now with molecular techniques as well.

 Heirtoruin mentions the works of Sean Carroll, he popularized the field of Evolutionary Development or Evo Devo and has discovered the "toolkit gene" in short, and this is just one example: the same genes are responisble for both antannae in insects as well as appendages in vertebrates.  He claims that old genes learn new tricks to drive evolution.  It's very interesting stuff.  For those of you who are not so science minded, I recommend picking up his book: The Making of the Fittest: DNA and the Ultimate Forensic Record of Evolution.  RRS had at one time, upon my request posted the book on the home page. But it has since been taken off.  I've been urging people to read Sean Carroll's books he's a very eloquent speaker and explains evolution in a matter where even the dumbest fundie can understand (he's friends with one of my thesis committee's professors) 

http://seanbcarroll.com/ 

 

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