Spotlights
ER stress: how trypanosomes deal with it Albrecht Bindereif and Christian Preußer Institute of Biochemistry, Justus Liebig University of Giessen, D-35392 Giessen, Germany
An efficient response to endoplasmic reticulum (ER) stress is essential for the viability of eukaryotic cells. The causative agent of African sleeping sickness, Trypanosoma brucei, responds to such stress by inducing spliced leader RNA silencing (SLS), resulting in shutdown of mRNA biogenesis. A new study elucidates the activation cascade and its molecular components, which are unique to the ER stress response in trypanosomes. The response to all kinds of cellular stress requires strictly controlled regulatory mechanisms. For example, in the endoplasmic reticulum (ER), where protein complexes are assembled and modified, stress can be caused by the accumulation of misfolded or unfolded proteins. The unfolded protein response represents a stress reaction particularly well-characterized in yeast and higher eukaryotic cells. Various regulatory processes are interconnected, ultimately provoking programmed cell death, also referred to as apoptosis. In metazoans three ER transmembrane proteins are involved in this response as stress sensors: the inositol-requiring enzyme 1 (IRE1), the protein kinase R-like endoplasmic reticulum kinase (PERK), and the activating transcription factor 6 (ATF6). Each factor is integrated in a separate, sophisticated response mechanism. Activation of IRE1 leads to the degradation of ER-associated mRNAs, ATF6 is a transcription activator for various stress-relevant genes encoding for example chaperones, and PERK inhibits the translation of other proteins by phosphorylating eukaryotic initiation factor 2 a (eIF2a) [1]. In contrast to other eukaryotes, unicellular parasites are peculiar in many respects of their cellular and molecular biology. A recent study by Hope et al. [2], based on the Trypanosoma brucei system, exemplifies this again for the response mechanisms to cellular stress signals, which appear divergent and rather simplified in these parasites. Trypanosomatids lack the conventional transcriptional regulation of protein-coding genes. Therefore it was not surprising to find that classical transcriptional mechanisms of the unfolded protein response, such as the IRE1-mediated response, are absent in trypanosomatids. This also holds for other unicellular parasites, for example Plasmodium and Toxoplasma [3]. In trypanosomes the stress response is mainly mediated by stabilizing mRNAs, which are essential Corresponding author: Bindereif, A. ([email protected]) Keywords: Trypanosoma; unfolded protein response; SL RNA silencing; programmed cell death. 1471-4922/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pt.2014.10.006
for survival on persistent stress [4]. In the closely related genus Leishmania and in Trypanosoma cruzi, the PERKmediated activation cascade had previously been found as an additional ER stress response pathway [3,5]. As an alternative way of stress response unique to T. brucei, the spliced leader silencing (SLS) pathway, leading to a complete repression of SL RNA gene transcription, had been described by the Michaeli group [4,6]. Why is this of special interest? In trypanosomes the SL RNA is essential for the expression of protein-coding genes in general, since – via trans splicing – the SL RNA provides its 50 terminal leader (miniexon) and cap structure to every mRNA. The SL RNA-encoding genes are the only ones in trypanosomes so far, for which a defined RNA polymerase II promoter could be identified. Transcription of the SL RNA genes requires the assembly of a pre-initiation complex, consisting of the small nuclear RNA-activating protein complex (SNAPc), the trypanosomatid homolog of the TATA-box binding protein, TRF4, and other transcription factors on a promotor element approximately 60 nucleotides upstream of the transcription start site [7]. In their recent study [2], Hope and colleagues reveal the first insight into the molecular mechanism of how the SLS mechanism is activated and regulated upon persistent ER stress. The authors were able to show here that in T. brucei the putative PERK paralog eIF2K3, briefly named PK3, is required for the activation of the SLS pathway. They further showed that ER stress, as during the accumulation of unfolded proteins or after a pH drop, induces the phosphorylation of PK3. Interestingly, in contrast to PERK in other eukaryotes, T. brucei PK3 lacks a signal peptide and a transmembrane domain, suggesting that PK3 might localize at the surface of the ER. Phosphorylation of PK3 leads to its relocation from the ER to the nucleus. Inside the nucleus, a constituent of the SL transcription pre-initiation complex, namely TRF4, becomes phosphorylated at a single residue, Ser35. This again points to the uniqueness of the SLS pathway for T. brucei, as this phosphorylated serine is apparently not conserved within the trypanosomatid lineage. Finally, phosphorylation of TRF4 promotes the disassembly of the pre-initiation complex from the SL RNA promoter, which was convincingly visualized by a diffuse nuclear distribution of the transcription factors involved. By a classical annexin assay, in which annexin interacts with the plasma membrane phospholipid phosphatidylserine, a marker of early apoptosis, the authors nicely demonstrated that PK3 is essential for the induction of programmed cell death. In sum this study makes an important contribution to promote our understanding of the apoptotic-like pathways in unicellular parasites (Figure 1). Trends in Parasitology, December 2014, Vol. 30, No. 12
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Spotlights
Trends in Parasitology December 2014, Vol. 30, No. 12
pH
O2
DTT
Heat
Ca2+
Cell density
Sec71 Sec61 Sec63
Drugs
ER stress
ER PK3
mRNA stabilizaon
P PK3
SNAPc TFIIA
TRF4
SL
? Nucleus
SNAPc TFIIA
TRF4
P
SL trans splicing
RNA POL II Programmed cell death
TRENDS in Parasitology
Figure 1. Endoplasmic reticulum (ER) stress and the spliced leader silencing (SLS) pathway in Trypanosoma brucei. Upon stress – such as during pH drop, defects in translocation (Sec71/61/63), or dithiothreitol (DTT) treatment – misfolded proteins accumulate in the ER lumen, and the SLS pathway is induced. Through phosphorylation, the translocation of PK3 from the ER to the nucleus is triggered, followed by phosphorylation of the TATA-box binding protein TRF4. As a result, the pre-initiation complex including TFIIA disassembles at the SL RNA promotor, thereby inhibiting the recruitment of RNA Pol II. Subsequently mRNA biogenesis is shut off, ultimately leading to programmed cell death. In addition, certain mRNAs, which are essential for the response of persistent stress, are stabilized, most likely to ensure a proper induction of the SLS.
A final pivotal question arises, which has been debated for a long time [8–10]: why does a unicellular organism need a defined mechanism for programmed cell death at all? It appears that parasites place the common welfare of their population above the selfishness of a single individual. By controlling their clonal growth, the programmed cell death mechanism thereby allows unicellular parasites to increase their chances to survive in a hostile environment. Nothing else other than the well-known survival of the fittest principle applies here. Parasites might use stress response mechanisms like the SLS to eliminate the unfit individual cells, thus ensuring the survivability of the entire population. Tasks for future work will be to further elucidate the mechanisms of how programmed cell death is regulated in trypanosomes and how this interconnects with ER stress. For instance, how is the translocation of PK3 from the ER into the nucleus accomplished? Moreover, it is unclear whether PK3 performs the phosphorylation of TRF4 by itself or whether other factors are required. In addition, the lack of classical caspases in clades outside of metazoans suggests alternative ways of apoptosis, as discussed by Michaeli [10]. Further studies should unravel the full pathways of stress signal transduction in these unique parasites, an important research topic in need of further study. Ultimately, such molecular
550
peculiarities of trypanosomes should open up new therapeutic approaches to fight the devastating diseases caused by these parasites. References 1 Janssens, S. et al. (2014) Emerging functions of the unfolded protein response in immunity. Nat. Immunol. 15, 910–919 2 Hope, R. et al. (2014) Phosphorylation of the TATA-binding protein activates the spliced leader silencing pathway in Trypanosoma brucei. Sci. Signal. 7, ra85 3 Gosline, S.J. et al. (2011) Intracellular eukaryotic parasites have a distinct unfolded protein response. PLoS ONE 6, e19118 4 Goldshmidt, H. et al. (2010) Persistent ER stress induces the spliced leader RNA silencing pathway (SLS), leading to programmed cell death in Trypanosoma brucei. PLoS Pathog. 6, e1000731 5 Tonelli, R.R. et al. (2011) Protein synthesis attenuation by phosphorylation of eIF2a is required for the differentiation of Trypanosoma cruzi into infective forms. PLoS ONE 6, e27904 6 Lustig, Y. et al. (2007) Spliced-leader RNA silencing: a novel stressinduced mechanism in Trypanosoma brucei. EMBO Rep. 8, 408–413 7 Das, A. et al. (2008) RNA polymerase transcription machinery in trypanosomes. Eukaryot Cell 7, 429–434 8 van Zandbergen, G. et al. (2010) Programmed cell death in unicellular parasites: a prerequisite for sustained infection? Trends Parasitol. 26, 477–483 9 Proto, W.R. et al. (2013) Cell death in parasitic protozoa: regulated or incidental? Nat. Rev. Microbiol. 11, 58–66 10 Michaeli, S. (2012) Spliced leader RNA silencing (SLS) - a programmed cell death pathway in Trypanosoma brucei that is induced upon ER stress. Parasit. Vectors 5, 107
ER stress: how trypanosomes deal with it Albrecht Bindereif and Christian Preußer Institute of Biochemistry, Justus Liebig University of Giessen, D-35392 Giessen, Germany
An efficient response to endoplasmic reticulum (ER) stress is essential for the viability of eukaryotic cells. The causative agent of African sleeping sickness, Trypanosoma brucei, responds to such stress by inducing spliced leader RNA silencing (SLS), resulting in shutdown of mRNA biogenesis. A new study elucidates the activation cascade and its molecular components, which are unique to the ER stress response in trypanosomes. The response to all kinds of cellular stress requires strictly controlled regulatory mechanisms. For example, in the endoplasmic reticulum (ER), where protein complexes are assembled and modified, stress can be caused by the accumulation of misfolded or unfolded proteins. The unfolded protein response represents a stress reaction particularly well-characterized in yeast and higher eukaryotic cells. Various regulatory processes are interconnected, ultimately provoking programmed cell death, also referred to as apoptosis. In metazoans three ER transmembrane proteins are involved in this response as stress sensors: the inositol-requiring enzyme 1 (IRE1), the protein kinase R-like endoplasmic reticulum kinase (PERK), and the activating transcription factor 6 (ATF6). Each factor is integrated in a separate, sophisticated response mechanism. Activation of IRE1 leads to the degradation of ER-associated mRNAs, ATF6 is a transcription activator for various stress-relevant genes encoding for example chaperones, and PERK inhibits the translation of other proteins by phosphorylating eukaryotic initiation factor 2 a (eIF2a) [1]. In contrast to other eukaryotes, unicellular parasites are peculiar in many respects of their cellular and molecular biology. A recent study by Hope et al. [2], based on the Trypanosoma brucei system, exemplifies this again for the response mechanisms to cellular stress signals, which appear divergent and rather simplified in these parasites. Trypanosomatids lack the conventional transcriptional regulation of protein-coding genes. Therefore it was not surprising to find that classical transcriptional mechanisms of the unfolded protein response, such as the IRE1-mediated response, are absent in trypanosomatids. This also holds for other unicellular parasites, for example Plasmodium and Toxoplasma [3]. In trypanosomes the stress response is mainly mediated by stabilizing mRNAs, which are essential Corresponding author: Bindereif, A. ([email protected]) Keywords: Trypanosoma; unfolded protein response; SL RNA silencing; programmed cell death. 1471-4922/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pt.2014.10.006
for survival on persistent stress [4]. In the closely related genus Leishmania and in Trypanosoma cruzi, the PERKmediated activation cascade had previously been found as an additional ER stress response pathway [3,5]. As an alternative way of stress response unique to T. brucei, the spliced leader silencing (SLS) pathway, leading to a complete repression of SL RNA gene transcription, had been described by the Michaeli group [4,6]. Why is this of special interest? In trypanosomes the SL RNA is essential for the expression of protein-coding genes in general, since – via trans splicing – the SL RNA provides its 50 terminal leader (miniexon) and cap structure to every mRNA. The SL RNA-encoding genes are the only ones in trypanosomes so far, for which a defined RNA polymerase II promoter could be identified. Transcription of the SL RNA genes requires the assembly of a pre-initiation complex, consisting of the small nuclear RNA-activating protein complex (SNAPc), the trypanosomatid homolog of the TATA-box binding protein, TRF4, and other transcription factors on a promotor element approximately 60 nucleotides upstream of the transcription start site [7]. In their recent study [2], Hope and colleagues reveal the first insight into the molecular mechanism of how the SLS mechanism is activated and regulated upon persistent ER stress. The authors were able to show here that in T. brucei the putative PERK paralog eIF2K3, briefly named PK3, is required for the activation of the SLS pathway. They further showed that ER stress, as during the accumulation of unfolded proteins or after a pH drop, induces the phosphorylation of PK3. Interestingly, in contrast to PERK in other eukaryotes, T. brucei PK3 lacks a signal peptide and a transmembrane domain, suggesting that PK3 might localize at the surface of the ER. Phosphorylation of PK3 leads to its relocation from the ER to the nucleus. Inside the nucleus, a constituent of the SL transcription pre-initiation complex, namely TRF4, becomes phosphorylated at a single residue, Ser35. This again points to the uniqueness of the SLS pathway for T. brucei, as this phosphorylated serine is apparently not conserved within the trypanosomatid lineage. Finally, phosphorylation of TRF4 promotes the disassembly of the pre-initiation complex from the SL RNA promoter, which was convincingly visualized by a diffuse nuclear distribution of the transcription factors involved. By a classical annexin assay, in which annexin interacts with the plasma membrane phospholipid phosphatidylserine, a marker of early apoptosis, the authors nicely demonstrated that PK3 is essential for the induction of programmed cell death. In sum this study makes an important contribution to promote our understanding of the apoptotic-like pathways in unicellular parasites (Figure 1). Trends in Parasitology, December 2014, Vol. 30, No. 12
549
Spotlights
Trends in Parasitology December 2014, Vol. 30, No. 12
pH
O2
DTT
Heat
Ca2+
Cell density
Sec71 Sec61 Sec63
Drugs
ER stress
ER PK3
mRNA stabilizaon
P PK3
SNAPc TFIIA
TRF4
SL
? Nucleus
SNAPc TFIIA
TRF4
P
SL trans splicing
RNA POL II Programmed cell death
TRENDS in Parasitology
Figure 1. Endoplasmic reticulum (ER) stress and the spliced leader silencing (SLS) pathway in Trypanosoma brucei. Upon stress – such as during pH drop, defects in translocation (Sec71/61/63), or dithiothreitol (DTT) treatment – misfolded proteins accumulate in the ER lumen, and the SLS pathway is induced. Through phosphorylation, the translocation of PK3 from the ER to the nucleus is triggered, followed by phosphorylation of the TATA-box binding protein TRF4. As a result, the pre-initiation complex including TFIIA disassembles at the SL RNA promotor, thereby inhibiting the recruitment of RNA Pol II. Subsequently mRNA biogenesis is shut off, ultimately leading to programmed cell death. In addition, certain mRNAs, which are essential for the response of persistent stress, are stabilized, most likely to ensure a proper induction of the SLS.
A final pivotal question arises, which has been debated for a long time [8–10]: why does a unicellular organism need a defined mechanism for programmed cell death at all? It appears that parasites place the common welfare of their population above the selfishness of a single individual. By controlling their clonal growth, the programmed cell death mechanism thereby allows unicellular parasites to increase their chances to survive in a hostile environment. Nothing else other than the well-known survival of the fittest principle applies here. Parasites might use stress response mechanisms like the SLS to eliminate the unfit individual cells, thus ensuring the survivability of the entire population. Tasks for future work will be to further elucidate the mechanisms of how programmed cell death is regulated in trypanosomes and how this interconnects with ER stress. For instance, how is the translocation of PK3 from the ER into the nucleus accomplished? Moreover, it is unclear whether PK3 performs the phosphorylation of TRF4 by itself or whether other factors are required. In addition, the lack of classical caspases in clades outside of metazoans suggests alternative ways of apoptosis, as discussed by Michaeli [10]. Further studies should unravel the full pathways of stress signal transduction in these unique parasites, an important research topic in need of further study. Ultimately, such molecular
550
peculiarities of trypanosomes should open up new therapeutic approaches to fight the devastating diseases caused by these parasites. References 1 Janssens, S. et al. (2014) Emerging functions of the unfolded protein response in immunity. Nat. Immunol. 15, 910–919 2 Hope, R. et al. (2014) Phosphorylation of the TATA-binding protein activates the spliced leader silencing pathway in Trypanosoma brucei. Sci. Signal. 7, ra85 3 Gosline, S.J. et al. (2011) Intracellular eukaryotic parasites have a distinct unfolded protein response. PLoS ONE 6, e19118 4 Goldshmidt, H. et al. (2010) Persistent ER stress induces the spliced leader RNA silencing pathway (SLS), leading to programmed cell death in Trypanosoma brucei. PLoS Pathog. 6, e1000731 5 Tonelli, R.R. et al. (2011) Protein synthesis attenuation by phosphorylation of eIF2a is required for the differentiation of Trypanosoma cruzi into infective forms. PLoS ONE 6, e27904 6 Lustig, Y. et al. (2007) Spliced-leader RNA silencing: a novel stressinduced mechanism in Trypanosoma brucei. EMBO Rep. 8, 408–413 7 Das, A. et al. (2008) RNA polymerase transcription machinery in trypanosomes. Eukaryot Cell 7, 429–434 8 van Zandbergen, G. et al. (2010) Programmed cell death in unicellular parasites: a prerequisite for sustained infection? Trends Parasitol. 26, 477–483 9 Proto, W.R. et al. (2013) Cell death in parasitic protozoa: regulated or incidental? Nat. Rev. Microbiol. 11, 58–66 10 Michaeli, S. (2012) Spliced leader RNA silencing (SLS) - a programmed cell death pathway in Trypanosoma brucei that is induced upon ER stress. Parasit. Vectors 5, 107
