Evolution of Red Giants

REFERENCE: Charpinet et al. “A compact system of small planets around a former red-giant star.” Nature (2011) 480, pgs 496 – 499.    

Red giants are low to intermediate mass stars late in their lifetimes that have swelled to massive sizes.  It has been predicted that the Sun will become a red giant is 7.5 billion years at which time its radius will become 200 times larger than it is now.  Typically, planets that are orbiting a star at a radius less than 1 astronomical unit (AU) will be engulfed by the swelling red giant.  However, some post red giant stars still have giant planets orbiting them at radii much closer than 1 AU.

                Recent work by Telting and colleagues suggests both how planets can survive immersion in the red giant envelope and influence the evolution of the star.  KIC 05807616 (also known as KPD 1943+4058) is a B subdwarf star, which is much hotter and brighter than a typical subdwarf star and represents a rarer way a red giant star can evolve.  This type of subdwarf evolves when a red giant loses its outer envelope prematurely.  Charpinet et al. show that KIC 05807616 has two planets, which they name KOI 55.01 and KOI 55.02, slightly smaller than Earth still orbiting it.

                They authors further offer a reasonable scenario as to how the current system came to be.  They feel that both bodies were originally large, gaseous planets that were swallowed by the red-giant envelope.  The immersion triggered the red giant’s outer shell premature loss and evolution into a B subdwarf, however this process stripped the planets of their gaseous layers and left only the inner cores, which the authors see orbiting the star today.

                An alternative scenario involving the merging of two white dwarfs that resulted in planet formation followed by a secondary planet is possible, but highly unlikely.

Ebolaviruses

REFERENCE: Dias et al. “A shared structural solution for neutralizing ebolaviruses.” (2011) Nature Structural and Molecular Biology 18(12) pgs 1424 – 1427.

                Five types of ebolaviruses have been identified: Sudan, Ebola, Reston, Bundibugyo, and Tai Forest.  The Sudan and Ebola forms cause the predominant amount of human deaths and recently a new variant of the Sudan virus has been found in the Gulu district of Uganda.  While many monoclonal antibodies exist, only a handful can neutralize Ebola virus and none can neutralize Sudan virus.  In a recent Nature Structural and Molecular Biology publication, Dias et al. discuss their development of a neutralizing antibody for Sudan virus and a subsequent crystal structure of it bound to the Gulu-Sudan variant protein GP_1,2.

                GP_1,2 (glycoprotein) is a viral trimeric receptor solely responsible for bringing ebolavirus into a host cell.  The protein is so named because the entire amino acid sequence is expressed then cleaved to create GP₁ and GP₂.  However, these two proteins remain attached to each other via a disulfide bond until the viral membrane fuses with the endosomal membrane.   

                The monoclonal antibody developed here, referred to as 16F6, recognized native Sudan virus GP_1,2 and their work indicated that binding of 16F6 alone was enough to block infection.  The epitope for the antibody was revealed by X-ray crystallography to be at the base of the trimer (Figure 11.1).  Further work showed that Sudan virus could still attach to host cells and be internalized, which left the authors to speculate that the antibody is either inhibiting another unidentified factor or it is blocking an additional necessary conformational change in GP_1,2 that leads to successful infection.  

                The final sentence of their paper summarizes a possible far-reaching conclusion from their work as “...for viruses in general, successful immunotherapy and vaccine design may depend on targeting antibodies that anchor glycoprotein subunits together and prevent the conformational changes required for fusion.”

How to: Autophosphorylation

REFERENCES:

Malecka & Peterson. “Face-to-Face, Pak-to-Pak.” (2011) Structure 19(12), pgs 1723 – 1724.

Wang et al. “Structural Insights into the Autoactivation Mechanism of p21-Activated Protein Kinase.” (2011) Structure 19(12), pgs 1752 – 1761. 

                Many kinases require the phosphorylation of a residue within their active site to help maintain a conformation that is compatible with substrate binding/kinase activity.  While some kinases are able to phosphorylate other kinases, many times it is the kinase itself which performs autophosphorylation.  However, it is an interesting question: if the kinase requires phosphorylation to function but it itself must provide the phosphorylation, how does that work?  It is nearly a “chicken or the egg” problem.

                In the issue of Structure published on December 6^th, two papers discuss the de novo phosphorylation of the Pak1 (p21-activated kinase 1) protein.  A commentary offered by Malecka and Peterson discuss the history of common mutations often used by bench scientists to achieve “kinase-dead” proteins that are useful for crystallization as well as an overview of other kinases whose crystal structures reveal dimeric structures where the active loop of one subunit is placed within the active site of the other subunit.  The authors discuss two ways in which kinases can transiently adopt active conformations within the dimer to achieve autophosphorylation in trans: symmetric and asymmetric.  Symmetric structures, such as those seen for the kinases Chk2 and Ire1, place each others activation loops in their active sites while asymmetric structures, such as DAPK3 and IGF1R, have only one subunit place its activation loop within the active site of the other subunit.  

                Pak1, as reported by Wang et al, adopts a symmetric trans-autophosphorylation structure.  Interestingly, the authors also report that the common lysine mutations made within the active site are not 100% kinase-dead so caution should be used when trusting them as such.

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!

Prions

Weissmann et al. “Prions on the move.” (2011) EMBO Reports 12(11) pgs 1109 – 1117.

             

                Prions are the infectious agents responsible for Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy.  PrP^C is a 208 amino acid protein with two potential glycosylation sites.  Typically, it is found GPI-anchored to the plasma membrane outer surface.  PrP^SC is an aggregate of misfolded PrP molecules.  The aggregate recruits properly folded PrP to promote sequestration, protein misfolding and aggregate growth.

                A recent review by Browning and colleagues explores the recent literature to explain the leading theories on “barrier to transmission” and how prions can adapt to new environments.

                Consider a situation where the donor PrP is different in amino acid sequence from the recipient PrP.  Recipient PrP may have trouble joining the donor PrP aggregate for two reasons.  One, the differences in amino acid sequence may not allow the recipient PrP to adopt the necessary conformation needed for stable addition.  However, even when the PrP sequence is exactly the same, recipient PrP may still have problems, leading to the idea that different cellular environments and perhaps other proteins are involved in aggregate growth.

                An interesting study took 22L prions that could chronically infect PK1 cells in the presence of R33 cells and swainsonine.  Swainsonine is a small molecule that causes misglycosylation of proteins.  After forty population doublings, the prion population had become R33-incompetent and was sensitive to swainsonine.  When these new prions were placed back in the environment of the brain, the population changed back to being R33-competent and swainsonine insensitive.  

                The authors offer an excellent summation of these findings: “…a prion strain is a quasi-species, consisting of a major component and many variants, which are constantly being generated and selected against in a particular environment, as described earlier for RNA viruses and retroviruses.”  Comparing the adaptability of prions, a misfolded protein, to that of viruses, which bear genetic material and can respond to cellular changes with more plasticity, is fascinating.  The field strongly feels the changes in properties are most likely due to change in PrP^SC conformation.  

                Unfortunately, it also means that prions can develop drug resistance.  For this reason, many feel the best way to stop aggregate formation is to stop PrP synthesis or accelerate its turnover.  This idea has merit since PrP depletion in mice does not lead to devastating side effects.  However, the authors do end on the downer by saying that “no effective therapy is on the horizon.”

The Great Sirtuin Debate

REFERENCE: Burnett et al. “Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila.” Nature (2011) 477, pgs 482 – 485.

                It all started with a report that overexpression of SIR2 in budding yeast led to increased lifespan.  Follow up studies showed similar results in C. elegans and Drosophila, which lead researchers to pursue the relationship between calorie restriction (a known way to extend lifespan) and sirtuin expression.  These results bore resveratrol, a purported activator of human Sirtuin 1 (SirT1), which most of the general public will tell you is a component of red wine.  

                The dissent on the role of resveratrol and sirtuins in lifespan extension comes from labs at the University of Washington, University of Wisconsin, Amgen Inc., and Pfizer, among others.  Their papers explicitly say resveratrol has no activating properties on SirT1, conclusions which they back up with control studies using the Fleur-de-Lys system and crystal structures.

                Another blow to the importance of sirtuins came in a recent issue of Nature.    Burnett et al. studied C. elegans and Drosophila overexpressing sir-2.1 and closely accounted for the genetic backgrounds of each.  When taking into account these parameters, longevity increase was no longer noted.  Within fruit flies, the authors further concluded that dietary restriction did increase fly lifespan but was not dependent on Drosophila Sir2.  

Gem and colleagues stress the importance of “…controlling for genetic backgrounds and for the mutagenic effects of transgene insertions in studies on genetic effects on lifespan.”  As for the importance of sirtuins, they cannot support a strong relationship between sirtuins and lifespan extension.

Leaky Protons

REFERENCE: Alavian et al. “Bcl-X_L regulated metabolic efficiency of neurons through interaction with the mitochondrial F₁F₀ ATP synthase” Nature Cell Biology (2011) 13(10) pgs 1224 – 1233.

                B-cell lymphoma-extra large (Bcl-X_L), a member of the programmed cell death Bcl-2 family proteins, is the major anti-apoptotic protein in adult neurons.  When Bcl-X_L is overexpressed, mitochondria translocate to presynaptic sites, the number and size of synpases increase, and the overall mitochondrial biomass goes up.  Synaptic strengthening requires high metabolism but the exact involvement of Bcl-X_L in these events is not clear.

                In this month’s edition of Nature Cell Biology, Alavian et al. detail a role for Bcl-X_L in binding to F₁F₀ ATP synthase.  Previous subcellular localization studies have placed Bcl-X_L within the mitochondrial outer membrane, but new immunoelectron microscopy data published here supports various other studies that suggest the protein is at the mitochondrial inner membrane, as well.  Further analysis showed that Bcl-X_L was present with the F₁F₀ ATP synthase complex and endogenous Bcl-X_L was co-immunoprecipitated with the ß subunit of ATP synthase.

                To clarify the role of Bcl-X_L binding, the authors studied ATP hydrolysis by submitochondrial vesicles enriched with F₁F₀ ATP synthase protein complexes (Figure 6.1).  The experiment used the H⁺ fluorescent indicator ACMA, which is unable to be transported into the vesicles.  Upon ATP hydrolysis, fluorescence of ACMA dropped as H⁺ is pumped out of the buffer and into the vesicles by the F₁F₀ ATPase.  Treatment of the vesicles with proton pump inhibitors or compounds that create vesicle pores (and subsequent proton leaks) resulted in higher levels of fluorescence upon ATP hydrolysis.  Interestingly, these same results were also seen when vesicles were treated with Bcl-X_L inhibitors.

                The hypothesis that Bcl-X_L is acting at F₁F₀ ATPase to prevent proton leak was further supported by patch-clamp studies where the authors measured leak conductance.  Conductance dropped dramatically in the Bcl-X_L overexpressing vesicles when either ATP or ADP was added to the buffer.  Reducing the amount of Bcl-X_L in these vesicles by way of knockdown studies showed the conductance to be higher across these membranes.

                The authors also showed that in Bcl-X_L overexpressing neurons, the uptake of oxygen is lower but ATP levels are higher than in wild type neurons and that recombinant Bcl-X_L can directly increase the rate of ATP hydrolysis by F₁F₀ ATPase.  Taken together, Jonas and colleagues conclude that Bcl-X_L reduces proton leak during ATP synthesis, which thereby increases the neurons’ ATP synthesis efficiency and improves their metabolism.

Ancient Humans

REFERENCE

Rasmussen et al. “An Aboriginal Australian Genome Reveals Separate Human Dispersals into Asia.” Science (2011) 334 pgs 94 – 98. 

Two theories exist to explain the ancestry of Aboriginal Australians (Figure 5.1).  The first, called the Single-Dispersal model, claims that Africans split from Eurasians, which then became Europeans and Asians, which led to Aboriginal Australians.  Unfortunately, the split between Europeans and Asians is believed to have occurred 17,000 to 43,000 years ago but archeological data suggests that anatomically correct humans were in Australian around 50,000 years ago.

                The second model, called the Multi-Dispersal model, suggests that an earlier and perhaps independent dispersal occurred before the split between Africans and Eurasians.  

                To determine which model is correct, Rasmussen et al. sequenced the genomic and mitochondrial DNA from the hair of an early 20^th century Aboriginal male and detailed their findings in the most recent issue of Science.    They found that Aboriginal Australians shared significantly more derived alleles with Asians (Cambodian, Japanese, Han and Dai) than Europeans (French) and that Europeans shared more derived alleles with Asians than Aboriginal Australians. 

                The authors went on to sequence three Han Chinese genomes and used this data to support their conclusions that Aboriginal Australians split from African populations before Eurasians differentiated into Europeans and Asians, thus supporting a Mutliple-Dispersonal model.  Fitting well with archeological data, it was concluded that this split occurred 62,000 to 75,000 years ago while the European/Asian split was 25,000 to 38,000 years ago.

                Rasmussen et al. concede that making one Aboriginal Australian DNA sample representative of an entire population may not be entirely fair.  However, if true, Aboriginal Australians are the direct descendants of the first humans in Australia and “…likely have one of the oldest continuous population histories outside of sub-Saharan Africa today.”

E. coli Infection

Reference

Zhang et al. “A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.” Nature Chemical Biology (2011) 7, pgs 671 – 677

                For the Gram-negative bacterium Escherichia coli to successfully infect a victim following ingestion, it must survive a trip through the stomach and reach the small intestine.  Mammalian stomachs create a low pH environment to help break down incoming proteins from both food eaten and any accidently ingested pathogens.  Unfortunately, several enteric bacteria, including some strains of E. coli, are able to survive the acidic stomach to arrive at the neutral small intestine intact and successfully infect a victim.

                The outer membranes of Gram-negative bacteria are quite porous and will allow passage of molecules smaller than 600 Da.  Obviously protons can easily cross that membrane to reach the periplasmic proteins within.  How do the bacteria protect these proteins from either denaturation at low pH (stomach) or incorrect renaturation upon reaching neutral pH (small intestine)?

                It was previously known that the bacterial protein HdeA binds periplasmic bacterial proteins at low pH to protect them.  Once reaching the neutral small intestine, HdeA releases its substrates in a nonactive form that must then be properly folded again for full function.  What additional chaperones were involved in this process as well as substrates for HdeA were unknown.

                Recent work by Chen and colleagues, published last month in the journal Nature Chemical Biology, focused on identifying substrates for HdeA by using an unnatural amino acid (named DiZPK by the authors) whose side chain can photocrosslink with proximal protein.  They were able to place this version of HdeA inside living E. coli cells, subject them to low pH and thus identify substrates for HdeA.

                Interestingly, the two substrate proteins identified here are DegP and SurA, both of which are essential chaperone protein themselves.  The authors theorize that HdeA exists to protect these two important chaperones at low pH and helps refold them upon neutralization, which means they are then subsequently free to help other proteins refold (Figure 4.1, directly from their paper).  While the cytosol has mechanisms in place for chaperone protein folding mediated by ATP, the periplasmic space is low in ATP so the bacteria have developed another way to circumvent the situation.

                The importance of HdeA could lead to new therapies to treat E. coli infections.  

VEEV

Reference: Zhang et al. “4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus” EMBO (2011) 30(18), pgs 3854 – 3863.

VEEV = Venezuelan equine encephalitis virus

Fast Facts

-                Capable of infecting both humans and all species of equine (horses, zebras, donkeys)

-                Mosquito-borne pathogen

No human vaccine or antivirual drugs are available to treat VEEV.  Instead, an attenuated virus exists known as TC-83, which is given to laboratory workers and military personnel as a vaccine.  Because of its inability to be treated, high infection rate, and ease of production, VEEV has the potential to be used in bioterrorism.  In fact, the United States and a few other countries have developed VEEV as a biological weapon.  

In a recent issue of The EMBO Journal, Zhang et al. published the 4.4 Å electron cryo-microscopy structure of TC-83.  Partial X-ray crystallography structures were known of the viral coat proteins E1 and E2, but this data allowed researchers to determine reasonable models for both entire proteins.  

Figures 3.1 and 3.2 are taken directly from the paper and show the reported structure for one viral particle in 3D and a cross section of the virus.

Researchers say their data partially explains why TC-83 is attenuated compared with other VEEVs and offers insights on host recognition and initial nucleocapsid core formation.  For a virus we need to understand better, this structure and their work is definitely a step forward. 

The reference is above if you’d like to read more!

Dimers, Tetramers, and DNA – Oh my.

REFERENCE:  Aramayo et al. Nucleic Acids Research (2011) 
                        Epub ahead of print: July 14^th, 2011

The tumor suppressor p53 transactivates genes involved in cell cycle arrest, apoptosis or senescence.  Several key papers have established p53’s structure as a dimer of dimers.  The central core domain, where the majority of cancerous mutations reside, is responsible for sequence specific binding to DNA.  Despite years of structural work, several questions, including the spatial arrangement of all p53’s domains and the basis for dominant negative p53 mutant effects, remain.  A recent paper by Aramayo et al. discusses a 21 Å cryo electron microscopy structure where full length murine p53 is bound to DNA duplexes bearing consecutive p53 recognition elements (REs).  Their data suggests that only one core domain of each dimer binds to an RE while the other two core domains remain unoccupied.  This type of complex requires a ~ 45° rotation of one dimer relative to the other upon DNA binding, a movement which is notable in other DNA binding proteins.  This rotation also allows for the C terminal domains, known to bind DNA nonspecifically, to come in contact with the duplex, while the N terminal domains become poised for interaction with the replication machinery.  It was previously established that p53 dimers are formed co-translationally while tetramers are formed post-translationally.  One allele bearing a core domain mutation will lead to dimers where each subunit bears the mutation.  This information, along with the described structure, offers a mechanistic explanation for why one mutated allele leads to dominant negative effects in p53 function: with one mutated allele, 75% of the p53 tetramers will be unable to bind DNA.

Double Duty Inhiitor?

REFERENCE: Hansen et al. Structure (2011) 19, pgs 919 – 929.

Malaria, a disease that causes 1 million deaths per year, is caused by a Plasmodium parasite.  Cysteine proteases (CPs) expressed by the parasite are implicated in key process of both parasitic life stages: liver and blood.  Interestingly, host cell CPs are also integral to infection.  Given the destructive nature of proteases, CPs of both host cells and parasites must be regulated site-specifically and temporarily.  In the July issue of Structure, Hilgenfeld and colleagues discuss the structure of the Plasmodium cysteine protease falcipain-2 (FP-2) in complex with the C terminus of their identified CP inhibitor from Plasmodium berghei (PblCP-C).  PblCP-C has an Ig-like ß sandwich fold and its closest structural relative is identified as chagasin, an I42 inhibitor family member.  Loops L0, L2, L4, and L6 protrude from PblCP-C (shown) into the active site of FP-2, thus occluding substrate binding.  The authors compare the PblCP-C:FP-2 structure to other solved inhibitor complexes and conclude that the major interactions responsible for inhibition are conserved between the structurally unrelated inhibitors, but the PblCP-C L0 interactions with FP-2 are unique to this complex.  Intriguingly, the structure of L0 also explains why PblCP-C is a potent inhibitor of the papain-like protease cathepsin L but not cathepsin B.  Because PblCP is necessary for host cell invasion, it is postulated that this CP inhibitor could block potentially deleterious protease activity at crucial moments, such as host-cell invasion, or inhibit host cell CPs (such as the cathepsin-like caspases).  It also provides a framework for developing small molecule inhibitors of the critically important FP-2.

Welcome!

Welcome to my new spin off blog!

My original intention for the blog Amedeo was to explain science topics to those who don’t have a strong scientific background.  I’m proud to say that blog is booming and readership is growing.  I truly enjoy writing those weekly posts and it makes me so happy to hear “I understand what you’re talking about!”  Science isn’t a mystery – I swear.

However, other readers out there have stronger scientific background and, like me, probably don’t have a tremendous amount of time to read the newest literature.  In between running experiments, dealing with everyday life, or being buried with work, the latest breakthroughs on vaccines or recent models for protein function are simply overlooked.

As a member of AAAS (The American Association for the Advancement of Science), the journal Science is emailed to me each week.  I usually only spend a few moments reading the headlines before moving on to other emails or other interests.  I know I’m not alone in that behavior and I very much want to change it!  This blog will now allow me to practice writing short reviews of interesting science papers as well as give my readers an idea of what is currently being published.  

I will be posting in weekly installments with new posts appearing on Saturdays or Sundays, the same as with Amedeo.  However, I’ll amend how I post based on how things are going.  Stay tuned as I learn to juggle two blogs!  And, as always, check back for new updates, layouts, or pictures as I tend to work on the blogs most days even if a new post hasn’t appeared.  They are constantly being updated!