Category Archives: Articles Review

It’s just take take take with these GAS: Bacterial modulation of host cell metabolism

Infections generally follow a similar path with some consistent steps. The microbe gains entry to the host, replicates in the host and is shed, in order to spread to a new host. Commonly this has the unfortunate side effect of making the host a bit ill.

For bacteria, replication within a host relies upon, among other things (such as avoiding the murderous cells of the host immune system), the ability of the microbe to obtain essential nutrients from the environment it finds itself in. A study published in Cell now shows that the pathogenic bacterium, group A Streptococcus (GAS), uses a mechanism that directly modulates the metabolism of the host cells in order to stimulate its own replication and proliferation.

GAS causes a variety of human infections and in fact only infects humans. Causing the well-known “strep throat” the majority of illnesses involving this bacterium are relatively mild. This is because the bacterium is commonly only found on the skin or in the throat. If this changes and the bacterium finds its way into more internal tissues, the blood or lungs for example, severe disease such as necrotising fasciitis and streptococcal toxic shock syndrome can result. Worldwide there are 700 million cases of mild GAS infection with about 650,000 cases becoming severe invasive infections. These 650,000 cases are associated with a mortality rate of around 25%.

Upon entering the host it is important for the bacteria to quickly gather nutrients in order to proliferate and truly establish itself at the site of infection. GAS attaches to host cells and releases the toxins streptolysin O (SLO) and streptolysin S (SLS) into the host cell. These toxins stimulate endoplasmic reticulum (ER) stress within the host cell. ER stress causes an increased unfolded protein response, ER-associated protein degradation (ERAD) and eventually cell death through a variety of pathways. It is associated with a medley of diseases such as diabetes and Alzheimer’s disease. In this case the change we are interested in is the increase in production of the enzyme asparagine synthetase that catalyses the production of asparagine (ASN). The researchers found that the host cell secretes increased levels of ASN which are detected by the bacterium, causing a sweeping change in gene expression that affects nearly 17% of the bacterium’s genes. These changes in gene expression include the up-regulation of genes involved in proliferation. In the absence of ASN these genes were down-regulated and the production of SLS/SLO was increased.

Graphical representation of the described pathway Image credit: Cell
Graphical representation of the described pathway
Image credit: Cell

This mechanism was only active locally and temporarily upon initial attachment, implying that this is a mechanism used by GAS to establish infection early on. Interestingly the detection of the increased ASN uses the two component system TrxSR, which is heavily involved in regulation of the bacterium’s virulence and metabolic genes. The researchers claim that this demonstrates that this pathway is an “important attribute to GAS pathogenesis” and helps the bacterium to cause severe disease. Other bacterial pathogens have been reported to benefit from host cell metabolism modulation, such as Agrobacterium tumefaciens, which makes plant cells produce opines required by the bacterium. The scientists point out that there are other pathogenic bacteria that use SLS/SLO-like toxins such as S.aureus and L.monocytogenes (both known for their ability to ruin a good takeaway or cream cake) and that it would be interesting to see if they use similar pathways. 

Reconciling conflicting results in Alzheimer’s disease research

VerghesevCarloAmyloid beta (Aß) deposition is a hallmark pathology in Alzheimer’s disease (AD). The apolipoprotein E (ApoE) gene, which encodes for the ApoE protein, has been established as a strong influence for the development of late-onset AD(1). The link between ApoE and Aß is unclear. One theory postulates that ApoE directly binds with Aß to mediate Aß clearance(2-4), while a second theory suggests the effects of ApoE on Aß clearance are indirect(5-7). Recent papers by Carlo et al(8) and Verghese et al(9) support these two respective contradicting theories.

In line with the first theory of a direct interaction between ApoE and Aß, Carlo et al found that sortilin, and not low-density lipoprotein receptor-related protein 1 (LRP1), mediates the cellular uptake of Aß/ApoE complexes, suggesting that sortilin is a major ApoE receptor that is essential for Aß clearance(8). In contrast, Verghese et al demonstrated that ApoE and Aß rarely bind together and that ApoE competes with Aß to bind with LRP1(9), which plays a role in neuronal Aß uptake(10). Thus, Verghese et al’s results suggest that ApoE inhibits Aß clearance via binding to LRP1(9). Verghese et al’s findings therefore are in accordance with the second theory that ApoE indirectly affects Aß clearance.

It would appear as though the experiments done by Carlo et al and Verghese et al are in contradiction with each other. However, there are important differences between these studies that may contribute to their contrasting results. First, Carlo et al examined the effects of sortilin and LRP1 on Aß/ApoE complexes, while Verghese et al did not use Aß/ApoE complexes in their experiments with LRP1. This might indicate that LRP1 is capable of binding to Aß only when it is not bound to ApoE. While there is an overwhelming amount of evidence that shows a direct interaction between ApoE and Aß(11-16), which supports Carlo et al, Verghese et al pointed out that the majority of studies demonstrating Aß/ApoE binding used synthetically prepared Aß at above physiological concentrations and did not assess the mechanisms behind the effects of Aß/ApoE complexes on Aß metabolism9. Verghese et al challenges previous literature as they show that Aß and ApoE rarely bind together under physiological conditions yet ApoE continues to affect Aß clearance, suggesting that the primary role of ApoE in Aß clearance is unlikely due to ApoE sequestering Aß. This implies that the two groups examined different pathways of ApoE-related Aß clearance: one pathway that depends on Aß/ApoE complexes and one that does not. Thus, the results from the two groups do not necessarily oppose each other as they targeted different pathways that may both contribute to Aß clearance, but the question remains as to which pathway contributes the most to Aß clearance.

In order to reconcile the results from Carlo et al and Verghese et al, several experiments could be done. Aß uptake should be measured in sortilin-expressing, sortilin-deficient, LRP1-expressing, and LRP1- deficient cells incubated with soluble Aß only, soluble Aß with ApoE (with molar ratios in the physiological range, as previously described(9)), and Aß complexed with ApoE (as previously described(8)). This would enable us to determine how free ApoE and Aß-bound ApoE influence Aß clearance with and without sortilin and LRP1. This would also enable us to see whether sortilin is capable of clearing Aß when it is not complexed with ApoE and conversely, whether LRP1 is capable of clearing Aß when complexed with ApoE. These experiments may clarify which pathway has the largest influence on Aß clearance. ApoE expression can be controlled by LXRs(17). Therefore, upregulating and downregulating the expression of ApoE with LXR agonists and antagonists in amyloid mouse models to examine the effects on Aß clearance may also be useful. If Aß clearance is increased by ApoE upregulation, then that would support Carlo et al’s finding that ApoE binding to Aß is essential for the latter’s clearance. If Aß clearance is increased by ApoE downregulation, then that would support Verghese et al’s finding that ApoE inhibits Aß clearance. In fact, there have been conflicting results regarding the effects of increasing or decreasing ApoE expression, as upregulating ApoE has been shown to facilitate Aß clearance(18), yet reduced ApoE expression may reduce Aß levels(19). Therefore, modifying ApoE expression in sortilin-expressing, sortilin knock-outs, LRP1- expressing, and LRP1-knock-outs crossed with amyloid mice could also be done to see how these receptors impact Aß clearance depending on the level of ApoE expression.

References

1 Poirier, J. et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 342, 697-699, doi:0140-6736(93)91705-Q [pii] (1993).

2 Koistinaho, M. et al. Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides. Nat Med 10, 719-726, doi:10.1038/nm1058nm1058 [pii] (2004).

3 Morikawa, M. et al. Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-beta. Neurobiol Dis 19, 66-76, doi:S0969-9961(04)00279-7 [pii]10.1016/j.nbd.2004.11.005 (2005).

4 Jiang, Q. et al. ApoE promotes the proteolytic degradation of Abeta. Neuron 58, 681- 693, doi:10.1016/j.neuron.2008.04.010S0896-6273(08)00328-0 [pii] (2008). 5 Kim, J. et al. Overexpression of low-density lipoprotein receptor in the brain

markedly inhibits amyloid deposition and increases extracellular A beta clearance. Neuron 64, 632-644, doi:10.1016/j.neuron.2009.11.013S0896-6273(09)00896-4 [pii] (2009).

6 Basak, J. M., Verghese, P. B., Yoon, H., Kim, J. & Holtzman, D. M. Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Abeta uptake and degradation by astrocytes. J Biol Chem 287, 13959-13971, doi:10.1074/jbc.M111.288746M111.288746 [pii] (2012).

7 Katsouri, L. & Georgopoulos, S. Lack of LDL receptor enhances amyloid deposition and decreases glial response in an Alzheimer’s disease mouse model. PLoS One 6, e21880, doi:10.1371/journal.pone.0021880PONE-D-11-05741 [pii] (2011).

8 Carlo, A. S. et al. The pro-neurotrophin receptor sortilin is a major neuronal apolipoprotein E receptor for catabolism of amyloid-beta peptide in the brain. J Neurosci 33, 358-370, doi:10.1523/JNEUROSCI.2425-12.201333/1/358 [pii] (2013).

9 V erghese, P . B. et al. ApoE influences amyloid-beta (Abeta) clearance despite minimal apoE/Abeta association in physiological conditions. Proc Natl Acad Sci U S A 110, E1807-1816, doi:10.1073/pnas.12204841101220484110 [pii] (2013).

10 Kanekiyo, T. et al. Heparan sulphate proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal amyloid-beta uptake. J Neurosci 31, 1644-1651, doi:10.1523/JNEUROSCI.5491-10.201131/5/1644 [pii] (2011).

11 Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E. & Ikeda, K. Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer’s disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain Res 541, 163-166, doi:0006-8993(91)91092-F [pii] (1991).

12 Strittmatter, W. J. et al. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A 90, 1977-1981 (1993).

13 Bales, K. R. et al. Apolipoprotein E is essential for amyloid deposition in the APP(V717F) transgenic mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 96, 15233-15238 (1999).

14 LaDu, M. J. et al. Isoform-specific binding of apolipoprotein E to beta-amyloid. J Biol Chem 269, 23403-23406 (1994).

NEUR 602: Carlo et al vs Verghese et al Angela Tam

15 Yang, D. S., Smith, J. D., Zhou, Z., Gandy, S. E. & Martins, R. N. Characterization of the binding of amyloid-beta peptide to cell culture-derived native apolipoprotein E2, E3, and E4 isoforms and to isoforms from human plasma. J Neurochem 68, 721- 725 (1997).

16 Kim, J., Basak, J. M. & Holtzman, D. M. The role of apolipoprotein E in Alzheimer’s disease. Neuron 63, 287-303, doi:10.1016/j.neuron.2009.06.026S0896- 6273(09)00549-2 [pii] (2009).

17 Laffitte, B. A. et al. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci U S A 98, 507-512, doi:10.1073/pnas.021488798021488798 [pii] (2001).

18 Riddell, D. R. et al. The LXR agonist TO901317 selectively lowers hippocampal Abeta42 and improves memory in the Tg2576 mouse model of Alzheimer’s disease. Mol Cell Neurosci 34, 621-628, doi:S1044-7431(07)00021-8 [pii]10.1016/j.mcn.2007.01.011 (2007).

19 Bien-Ly, N., Gillespie, A. K., Walker, D., Yoon, S. Y. & Huang, Y. Reducing human apolipoprotein E levels attenuates age-dependent Abeta accumulation in mutant human amyloid precursor protein transgenic mice. J Neurosci 32, 4803-4811, doi:10.1523/JNEUROSCI.0033-12.201232/14/4803 [pii] (2012).

REVIEW of Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains (Puthenveedu et al. 2011)

Endosome is an important sorting compartment, as it mediates biosynthetic and endocytic pathways, including bulk recycling, lysosomal degradation and sequence-dependent sorting. In this paper, Puthenveedu hypothesize that the sequence-dependent pathway uses distinctive features, such as local enrichment of cargo and cytoskeleton. These features allow sorting-sequence-containing receptors to escape from conventional pathways. The sequence-dependent pathway is newly emerged. As the mechanism of this novel pathway remains fragmentary, this paper is remarkable as it shed light on how some receptors are recycled in a regulated fashion.

Puthenveedu approached the hypothesis by utilizing four major tools: (i) fluorescence microscopy; (ii) fluorescene recovery after photobleaching (FRAP); (iii) latrunculin treatment; and (iv) recombinant and mutant receptors. β 2-adrenergic receptor (B2AR) was used as a model as it is recycled to cell surface via sequence-dependent sorting pathway. First, various FLAG-tagged and GFP-tagged proteins were expressed in 293 cells to identify the localization of proteins. To trace the translocation of B2AR membrane receptors from endosome to cytosol, sGFP-B2AR was used. sGFP-B2AR was sensitive to pH as it fluoresces at neutral pH in cytosol but not in acidic endosomes or transporting vesicles. Next, FRAP was used to study the diffusion rate of receptors and the turnover rate of cytoskeleton by making a “dark box” to observe the movement of fluorescent molecules of interests. To study the role of actin polymerization, latrunculin was used to inhibit actin polymerization. Then, to confirm the importance of PDZ domain in the pathway, a B2AR mutant with Ala insertion in PDZ domain was used.

By utilizing the above techniques, B2AR is found to be enriched in the tubular domain on endosomes. The majority of B2AR is delivered to the cell surface using this tubular domain that is biochemically distinct from other domain for bulk recycling pathway. In particular, actin filaments were found to be concentrated at the B2AR-containing tubules. In the absence of endosomal actin filaments, less than 20% of endosomes have B2AR-containing tubules, suggesting the importance of actin in the sequence-dependent sorting pathway. To promote actin polymerization, Arp 2/3 complex enables the initial nucleation step. Interestingly, Arp 3 is found at the base of the B2AR-containing tubular domain. While some Arp 2/3 activators, N-WASP and WAVE-2, are absent from endosomes, WASH protein complex is localized with the B2AR-containing tubules and cortactin is localized at the base of the tubules. Furthermore, the PDZ domain of B2AR is important in promoting the formation of B2AR-containing tubules. Comparing to Tfr which is dependent on bulk flow recycling pathway, B2AR has a significantly lower diffusion rate than Tfr, and extrusion of B2AR-containing tubules is lower than Tfr tubules

Puthenveedu and his group have presented some convincing results as to how sequence-dependent sorting pathway works distinctively from the other conventional pathways. However, as “24.4% of Tfr tubules contained detectable B2AR”, I am not convinced as most experiments are qualitative and observation-based. The significant amount of B2AR found in Tfr tubules suggests that sequence-dependent sorting pathway goes parallel with bulk recycling or an unknown pathway. So, how can a significant amount of B2AR escape from the sequence-dependent sorting pathway? One possibility is that a protein is required to mediate the interaction of B2AR to the actin filament. Due to the limited quantity of this mediator protein, a constant flow of B2AR can be recycled back to the cell surface via the bulk recycling pathway. In other words, this potential mediator holds the key in regulating the sequence-dependent sorting pathway in response to cellular demand and environmental stimulus. To initiate the approach to validate my hypothesis, we need to sort out B2AR-containing endosomes, then capture B2AR and its interacting partner for mass spectroscopy to identify the potential mediator protein. To confirm the interaction, mammalian or yeast two-hybrid systems and gel shift can be performed.

Reference:

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains