Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.

An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei.

If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganisms on the cellular level. Here, we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4 -induced stresses share many common metabolic features, for example, signalling pathways, elevated energy metabolism, or osmolyte biosynthesis. Nevertheless, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for putative life on Mars, which-given the environmental conditions-likely developed chaotropic defence strategies such as stabilized confirmations of biomacromolecules or the formation of cell clusters. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1462-2920.16152

Perchlorate‐specific proteomic stress responses of <scp> <i>Debaryomyces hansenii</i> </scp> could enable microbial survival in Martian brines

Mitochondria are physically associated with other organelles, such as ER and lysosomes, forming a complex network that is crucial for cell homeostasis regulation. Inter-organelle relationships are finely regulated by both tether systems, which maintain physical proximity, and by signaling cues that induce the exchange of molecular information to regulate metabolism, Ca2+ homeostasis, redox state, nutrient availability, and proteostasis. The coordinated action of the organelles is engaged in the cellular integrated stress response. In any case, pathological conditions alter functional communication and efficient rescue pathway activation, leading to cell distress exacerbation and eventually cell death. Among these detrimental signals, misfolded protein accumulation and aggregation cause major damage to the cells, since defects in protein clearance systems worsen cell toxicity. A cause for protein aggregation is often a defective mitochondrial redox balance, and the ER freshly translated misfolded proteins and/or a deficient lysosome-mediated clearance system. All these features aggravate mitochondrial damage and enhance proteotoxic stress. This review aims to gather the current knowledge about the complex liaison between mitochondria, ER, and lysosomes in facing proteotoxic stress and protein aggregation, highlighting both causes and consequences. Particularly, specific focus will be pointed to cancer, a pathology in which inter-organelle relations in protein aggregation have been poorly investigated.

/pdf/mitochondria-inter-organelle-relationships-in-cancer-protein-1ui93mmx.pdf

Mitochondria inter-organelle relationships in cancer protein aggregation

If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganism on the cellular level. Here we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4-induced stresses share many common metabolic features, e.g., signaling pathways, elevated energy metabolism, or osmolyte biosynthesis. However, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for life on Mars, which - given the environmental conditions - likely developed chaotropic defense strategies such as stabilized confirmations of biomacromolecules and the formation of cell clusters.

Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines

/pdf/an-essential-kinetoplastid-specific-gdp-fuc-b-d-gal-a-1-2-2ozs65k30d.pdf

An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in Leishmania major [H. Guo et al., J. Biol. Chem. 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative "plasmid segregation" assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null ∆fut1 - background, established that FUT1 is essential. Similarly, "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes, respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly, a single rare ∆fut1 - segregant (∆fut1 s ) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 reexpression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 [G. Bandini et al., bioRxiv, https://www.biorxiv.org/content/10.1101/726117v2 (2021)] joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting, and their essentiality for normal growth and mitochondrial function renders it an attractive target for chemotherapy of these serious human pathogens.

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania.

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. Recently we showed that GDP-Fucose synthesis is essential, although fucosylated glycoconjugates have not been reported in Leishmania major. The Leishmania genome predicts at least five candidate fucosyltransferases, four of which appear targeted to the secretory pathway; SCA1 and SCA2 play a role in side-chain modifications of the abundant surface glycoconjugate lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it to have fucosyltransferase activity, with a relative broad substrate specificity including glycans and peptide substrates. A quantitative plasmid segregation assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null Δfut1- background established that FUT1 is essential. We used plasmid shuffling to confirm that both enzymatic activity and mitochondrial localization were essential for viability, comparing import-blocked or catalytically inactive enzymes respectively. Unexpectedly a single rare Δfut1s mutant was obtained, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (1) joins the eukaryotic O-GlcNAc transferases as one of the few glycosyltransferases acting within the mitochondrion. Current work is now oriented towards identifying the Leishmania fucosylated targets therein.

/pdf/a-broadly-active-fucosyltransferase-lmjfut1-whose-4n1dnbbk1c.pdf

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in parasitic Leishmania major

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. While GDP-Fucose synthesis is essential (Guo et al 2017), fucosylated glycoconjugates have not been reported in Leishmania major. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative ‘plasmid segregation’ assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null Δfut1- background, established that FUT1 is essential. Similarly “plasmid shuffling” confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly a single rare Δfut1-s segregant (Δfut1s) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (Bandini et al 2021) joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting and their essentiality renders it an attractive target for chemotherapy of these serious human pathogens. Significance Statement Abundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites including Leishmania. Through defining biosynthetic pathways we identified a fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical compartment for glycosyltransferases. FUT1 was essential for normal growth, requiring both mitochondrial localization and enzymatic activity. Loss of FUT1 in a unique segregant showed extensive mitochondrial defects. Enzymatic tests showed FUT1 could fucosylate glycan and peptide substrates in vitro, although as yet the native substrate is unknown. Trypanosomatid mitochondrial FUT1s may offer a facile system in the future for probing mitochondrial glycosylation in a setting uncomplicated by multiple isoforms targeted to diverse compartments, and its essentiality renders it an attractive target for chemotherapy of these deadly parasites.

/pdf/a-broadly-active-fucosyltransferase-lmjfut1-whose-23k42tx30y.pdf

Luciana Penha

Papers

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania.

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in parasitic Leishmania major

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania