Progeria Hope!

REFERENCE: Gordon et al. “Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome.” PNAS (2012) 109(41) pgs. 16666 – 166671

                Between pharmaceutical companies and academic laboratories, the amount of compounds and antibodies available that can treat any number of diseases is staggering.  Either through rational design (choosing to target a protein’s particular function, shape, size, etc) or from blind mining (high throughput screens looking for a particular effect from any possible molecule), scientists are constantly searching for drugs to treat diseases.  Sometimes a particular drug is developed, honed, and specialized to treat a specific disease but its capabilities fall short.  Such was the case for lonafarnib, a farnesyltransferase inhibitor (FTI) that was originally designed to inhibit hydrophobic membrane-anchoring of GTPases, particularly Ras, which is overactive in many different cancer types.  Researchers studying Hutchinson-Gilford progeria syndrome (HGPS, also called progeria) recently decided to repurpose this FTI to see if it could also help patients suffering this lightening fast aging disease.

                Progeria is incredibly rare: only seen in one of four million live births.  Its patients suffer from diseases of old age (stiff joints, loss of body fat, cardiovascular disease) when they are only children.  No treatment or cure currently exist and, for this reason, progeria is considered 100% fatal.  Most children do not live past the age of 13.

                A single base mutation in the LMNA gene, which encodes for the protein lamin A, causes the lamin A RNA to splice differently and therefore encode for a shorter protein, known as progerin.  Progerin lacks the proteolytic cleavage site necessary to remove its C terminal farnesyl group.  Unfortunately, this means that all copies remain membrane bound and cluster specifically on the inner nuclear membrane.  Scientists believe that progeria symptoms are, in part, due to the increased progerin concentration at the nuclear membrane.  Lonafarnib, however, can bind to farnesyltransferase target proteins and inhibit them from ever gaining a farnesyl group.  An excellent candidate for this would be the progerin protein.  Early success for progeria research showed progerin-expressing cells normalizing in structure and function following lonafarnib treatment.  Mouse models treated with the drug also display improved health.  For these reasons, Gordon et al. designed a two year initial clinical trial for lonafarnib beginning in 2007 and now they report the encouraging results.

                Twenty five children, which account for 75% of all progeria patients in the world, completed the trial.  Side effects were allegedly mild and included fatigue, nausea, anorexia, depressed serum hemoglobin, vomiting, and diarrhea.  However, several improvements in progeria-related factors were noted within the patients: increased rate of weight gain, improved cardiovascular stiffness, improved bone structure and improved audiological status.  Every child in the study improved in at least one of the listed areas.

                Aortic stiffness is an indicator of cardiovascular health.  Before treatment, the average aortic stiffness age among the progeria patients was 60 – 69 years.  Following treatment, the average dropped 40 – 49 years.  For a disease that ultimately kills via cardiovascular problems (heart attacks, strokes), these results are very encouraging.

                Interestingly, even healthy individuals express progerin and its production increases with age.  The authors speculate that aging treatments in the future might rely on FTIs should they find that progerin plays a major role in aging among non-progeria patients.

                 

Personal Cancer Treatment

Reference: Muller et al. “Passenger deletions generate therapeutic vulnerabilities in cancer.” Nature (2012) 488, pgs 337 – 341

Reference: Lehner and Park. “Exploiting collateral damage.” Nature (2012) 488, pgs 284 – 285 

Reference: Johnston, Iain. “The chaos within: Exploring noise in cellular biology.” Significance (2012) August, pgs 17 – 21 

                Innovative ways to specifically kill cancer cells within the context of a living human body are necessary.  The scientific community has plenty of ideas: Trojan horse proteins bearing chemotherapeutics, exploiting the nature of cancer cell surface receptors, intense high throughput studies to identify cancer-sensitive compounds and targeted therapies towards known oncoproteins.  Unfortunately, just as each human is unique, cancer is an all-encompassing term for hundreds of different diseases that each has their own set of complications to be overcome.  Forward thinking and ingenuity are keys to successful progression.  To this end, Muller et al. report in this week’s Nature magazine about an experimental design that identifies key vulnerabilities in cancer cells by highlighting what proteins are not present.

                Enolase is an essential enzyme necessary for the second to last step of glycolysis.  Three homologues of this enzyme exist with three different gene expression profiles: ENO1 is ubiquitous, ENO2 is restricted to neurons, and ENO3 is only in muscle.  It has been shown that invertebrates and mice carry several homologous genes that encode for proteins capable of doing each other’s jobs.  The beauty of this redundancy occurs when one of the genes is knocked out: the other proteins are able to pick up the slack and death isn’t an inevitable result.  In the case of enolase, both ENO1 and ENO2 are expressed in neural cells and both are capable of performing the same function.  But, imagine that one gene becomes mutated.  The cell would then have an unhealthy reliance on the other ENO gene.  Since the enzyme is essential, a blow to the other enolase homologue should result in death to that cell.

                Cancer cells have an unhealthy habit of collecting mutations.  The Cancer Genome Atlas Research Network has sought to study the genomes of cancer to establish what mutations have turned a once healthy cell into a feast of illness.  Gliobastomas are a type of brain tumor that affects glial cells, which expresses both ENO1 and ENO2, but is far more reliant on ENO1.  Interestingly, the lp36 locus, home of the ENO1 gene (among others) is often deleted in glioblastoma.  In theory, this should create an Achilles Heel out of ENO2. 

                Muller et al. began with two different cells lines: one expressed both ENO1 and ENO2 (ENO1 wild type) while the other only expressed ENO2 (ENO1-null).  Upon two independent shRNA-mediated knockdowns, only the cells only expressing ENO2 displayed marked inhibition of proliferation.  Wanting to further prove their concept, the authors then treated the cells with the enolase inhibitor phosphonoacetohydroamate (PHAH).  The compound displayed potent toxicity towards the ENO1-null cells and little impact on the ENO1 wild type cells.  Finally, PHAH was titrated into cells with varying degrees of both ENO1 and ENO2 expression.  Intriguingly, the data showed a direct relationship between the sensitivity of the cells to PHAH and their enolase activity profiles.  

                The lp36 locus contains other essential housekeeping proteins.  In addition to these cells being reliant on ENO2, they might also be unnaturally resting on other individual proteins whose homologues have been knocked out.  Determining what they are and inhibiting them as well could lead to even greater effectiveness at cancer cell-specific death.  The paper ends up with this thought: “By one estimate, 11% of all protein-coding genes in the human genome are deleted in human cancers.  Thus, given the large number of homozygous deletions across many different cancer types spanning many hundreds of genes, the model described here for [glioblastoma] should be applicable to the development of personalized treatments for many other cancer types.”

                While reading this paper, I kept thinking back to another article I read in the journal Significance concerning the random events and complete chaos that is the inside of a cell.  Take two cells with exactly the same genome.  Variability exists in the expression profiles of all genes between those two cells due to random chance.  In the Muller et al. example, one single cancer cell in a gliobastoma may express ENO2 at a much higher level than its neighboring cell, which means that Cell A will need more drug to kill it than Cell B even though both have the exact same genome.  But scientists don’t consider Cell A and Cell B, we consider whole cell populations and assume they are all acting identically.  One IC₅₀ value represents the cells in that particular plate at that particular time and is averaged with other cell populations at different times.  Eventually, scientists are looking at averages of averages.  We make broad assumptions on broad pieces of data that are based on what is happening right then.  But cancer isn’t static; tumors within a human body are an ever evolving entity that picks up more mutations and creates more roadblocks as time goes on.  Cancer is a many-headed hydra.  

Crystallography: phasing out heavy atom derivatives

REFERENCE: Liu et al. "Structures from Anomalous Diffraction of Native Biological Macromolecules." Science (2012) 336, pgs 1033 - 1037.                 

        The field of protein crystallography has partially pulled back the veil that lies between the macroscopic and microscopic worlds.  Coupling crystallography with X-ray diffraction yields structure models that allow scientists to consider the shape, surface, and, in short, the angstrom-length details of a protein.  Each spot on a diffraction pattern arises from a constructive interference event between X-ray waves within the protein crystal.  Amplitude and frequency information, two of the three variables that define a wave, are provided by these spots, but the final parameter of phase must be experimentally determined before a protein model can be built.  “Solving the phase problem” has thus far relied on soaking proteins in heavy atoms, use of experimental phases from a closely related protein, or incorporation of selenomethionine into the polypeptide chain.  Scientists would rather determine structures using crystals of native protein but such a method has thus far been unavailable.  In a recent study published in the journal Science, Liu et al. discuss their new technique that uses anomalous dispersion from native atoms within the protein to obtain phase information; the authors further support their technique’s worth by solving the structures of four different proteins that vary in size and subcellular localization.

                Anomalous dispersion relies on a heavy atom’s ability to absorb and emit X-rays.  When the wavelength of the X-rays approaches the characteristic emission wavelength of the atom, then the absorption drops off sharply giving rise to anomalous scattering.  With access to tunable synchrotron beamlines and protein crystals bearing heavy atoms, crystallographers using multiple anomalous dispersion (MAD) or single anomalous dispersion (SAD) can gather data sets at their heavy atom’s absorption edge to identify the heavy atoms’ locations and eventually provide the phases necessary to build a protein model.  Selenium (Z = 34) has traditionally been added to proteins to create anomalous signals, but obtaining phase information from crystals of native protein would be optimal. Intrinsic iron (Z = 26) has occasionally been sufficient for phase determination, but the next heaviest atom regularly found in proteins is sulfur (Z = 16).  .  

                Since 1981, 57 novel structures have been published that employed light atom (Z <20) SAD to determine phases.  In comparison, over 5000 structures have been deposited into the Protein Data Bank (PDB, www.pdb.org) in the past fifteen years that used heavy atom SAD.  This discrepancy arises from the low anomalous strength of light atoms, low amount of sulfurs per protein molecule, radiation damage, and diffuse scattering of the X-rays.

                Determined to overcome these complications, Liu et al. began with their previously reported technique that improved data from poorly diffracting selenomethione protein crystals.  To decrease radiation damage and increase signal to noise, a procedure was designed to merge data from multiple crystals.  Building on this, authors then optimized the X-ray beam energy based on knowledge that anomalous signal from light atoms increases with increasing wavelength.  Together, these two methods increased signal to noise and minimized radiation damage.  Finally, the crystal and beam path were placed inside a helium-filled cone and the beam size was matched to crystal size, which also boosted signal to noise and reduced incoherent X-ray scattering.

                Four test proteins were crystallized: netrin G2, TorT/TorS_s, HK9s and CysZ.  Three of the four contained at least 20 sulfur atoms, but HK9s only had three plus an additional chloride.  At least 5 crystals were used per protein.  The authors then define three criteria each crystal’s data set must meet before being merged with others: unit cell parameters less than 3σ, overall diffraction dissimilarity less than 5%, and the relative anomalous correlation coefficient greater than 35%.  This is an improved rubric from their previous work.  Of the 31 crystals screened, only one data set was removed after failing the above standards.  For each protein, data sets were added together one at a time and tested for structure determination.  With each successive data set, anomalous signal, resolution, and electron density maps improved, thus validating their procedures.

                As a way to demonstrate the versatility of light atom anomalous dispersion, the test proteins were diverse.  They ranged from127 to 1148 ordered residues, their crystal symmetries varied from monoclinic to tetragonal and resolutions are as high as 2.3 Å.  Netrin G2 and TorT/TorS_s were both previously unknown structures, while HK9s and netrin G2 were not amenable to previous structure determinations.  CysZ is a membrane protein, while the rest of soluble domains of membrane proteins.  Finally, TorT/TorS_s is reported to be at a complexity level exceeding 90% of the current PDB and its structure was determined here to reasonable resolution.  The authors do admit that the technique will benefit from improvements at beamlines, scaling and weighting procedures, but they do believe that multicrystal SAD phasing will be extremely useful for determining de novo structures of native proteins and nucleic acids.

Controversial Influenza Research

REFERENCE: Imai et al. "Experimental Adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets." Nature (2012), epub ahead of print.  LINK

Amedeo Post offering more background and editorials related to this work: LINK

                This week in Nature sees the publication of the long discussed influenza research paper from the University of Wisconsin-Madison lab of Yoshihiro Kawaoka.  In a recent editorial, Kawaoka urges for publication and further research in this area by saying “[s]ome people have argued that the risks of such studies…outweigh the benefits.  I counter that H5N1 viruses circulating in nature already pose a threat because influenza viruses mutate constantly and can cause pandemics with great losses of life.  …I believe it would irresponsible not to study the underlying mechanisms.”  This published paper focuses on the hemaglutinin (HA) protein from the H5N1 virus circulating primarily in Southeast Asia that retains specificity and virulence in birds.  To date, 578 humans have become infected with this virus after direct contact with infected animals.  340 have died, but human-to-human transmission has, so far, not been an issue, but the potential for a pandemic caused by an evolving H5N1 virus is still present.  Kawaoka and colleagues sought to determine what mutations within this H5 would allow the protein to bind human receptors, if an influenza virus bearing these mutations could both efficiently infect and transmit the virus among mammals, and finally if current vaccines/antiviral therapies would be useful against such a virus.

                Human cells of the respiratory tract display α2,6-linked sialic acid with galactose while avian tracts have α2,3 linkages.  As the main receptors for HA binding, the ability of an influenza virus that was specific for birds to infect humans means, in part, that the specificity for binding switched from α2,3 to α2,6.  The authors began by introducing random mutations into the H5 globular head region where receptor binding occurs.  Turkey red blood cells were treated with sialidase to remove α2,3 linked sialic acid and preferentially leave α2,6.  Viruses were generated bearing the mutated H5s and tested for their ability to bind treated turkey red blood cells.  The identified viruses were screened again for α2,6 binding to root out false-positives and identified HAs were then further tested in solid-phase binding experiments for α2,6-specificity.  In the end, an H5 bearing mutations at E119, V152, N224 and Q226 was identified as binding only α2,6 linkages.  The authors further confirmed that N224 and Q226 mutations were critical for the shift in specificity.

                The hemaglutinin from H1N1, which was isolated from a human patient, was replaced with an H5 bearing the appropriate N224 and Q226 mutations.  Ferrets were infected with this reassortant virus and found that, after a period of 6 days, a new mutation at position N158 was observed. Viruses bearing an H5 with this new additional mutation could replicate well in ferrets and were mildly transmissible between ferrets.  Intriguingly, the viruses were found to contain yet another mutation as position T318 after ferret infection.  A new H1N1 virus bearing a quadruple H5 mutant protein was found to be highly transmissible between ferrets.  No ferrets died as a result of infection with either virus.  Encouragingly, the authors also showed that a prototype H5N1 vaccine was reactive with the mutant viruses created here and the viruses were susceptible to a licensed NA inhibitor.

                Specific mutations at N224 and Q226 are mostly likely changing the binding pocket to accommodate α2,6 linkages; a specific N158 mutation removes a glycosylation site on H5 that could be improving transmissibility.  These three mutations destabilize the protein in an acidic environment but the T318 mutation returns that stability.  Membrane fusion of the influenza virus with the host cell occurs at low pH so stability at these [H⁺] is necessary.

                It should be noted that hemaglutinin, while highly involved, is only one protein involved in the virulence of an influenza virus.  Studies show that neuraminidase also plays a role.  It was also stressed that the remaining genes in the mutant virus came from H1N1, not the avian-virulent H5N1.  It’s possible that the remaining genes in the influenza also contribute to the virulence of a virus in new hosts.  The hope is that the amino acid mutations identified here will help those keeping an eye on current H5N1 viruses.  Should any of these mutations arise, they will have the tools to predict pandemic potential and, knowing that current therapies are effective against these viruses, that proper safeguards can be put in place quickly.  As a final thought, the authors realize that a pandemic virus may not even show these mutations and something else entirely, but the work has identified important areas of the HA protein that could and most likely will change as an influenza virus evolves from avian to mammal specificity.

 

American Society for Cell Biology Meeting

Denver, CO: December 3^rd (Saturday) – December 7^th (Wednesday)

REFERENCE:  Science (2011) 334 pgs 1046 - 1051

                This Saturday marks the beginning of the annual ASCB meeting in Denver, CO.  The event will feature over 3000 scientific poster presentations as well as 100 scientific sessions.  Principal Investigators, post doctoral associates and graduate students from all over the world will descend on Denver to discuss science (and ski) starting this weekend.

                As a preview to this event, Science magazine published a five article series discussing some of the most pressing questions currently facing cell biologists.  They include…

Do lipid rafts exist?  This is a contentious topic, but important to understand.  The plasma membrane is the first line of defense for a cell and acts as a gate keeper to all the comings and goings.  Understanding how it works is essential.

How does a cell know its size?  Many different kinds of cells exist but all of them stay within a certain size.  Certain proteins have been identified in yeast and bacteria that are involved in cells “sensing” their size but more work needs to be done.

How does a cell position its proteins?  Some cells make upwards of 10,000 proteins.  Positioning all of them so that they are in the proper places to perform their functions efficiently is a monumental task.  Proteins carry targeting sequences that place them in different organelles, but new research suggests that mRNAs may also be playing a role.

How do hungry cells start eating themselves? Autophagy is becoming a hot topic!

Does a gene’s location in the nucleus matter? Nuclear organization is important to cell function.  In fact, in cancer and other diseases, the nucleus is reorganized.  Researchers are trying to understand why the cell likes its proteins and RNA in certain places relative to its chromosomes.

I highly recommend reading them.  The articles are short and already written in a summary format.  I don’t want to write a redux of a redux on this blog because that is ridiculous.  Instead, I implore you to pick up a November 25^th copy of Science magazine and read pages 1046 – 1051!