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!