11
Cell News 2/2015
in turn sequester unfolded proteins to support their folding. In
addition to chaperone assistance, successful protein folding
in the ER requires specific glycosylation of polypeptide chains,
which, in addition to being structural components of the protein,
also reflect its folding state. In case all attempts to fold a nascent
protein in its natural conformation fail, unfolded proteins are
degraded by the proteasome or through the autophagic pathway.
Naturally, the ER is particularly susceptible to proteotoxic stress
as it is a major site of protein synthesis and folding (Schröder
and Kaufman, 2005). The UPR
ER
can be activated through three
distinct branches allowing cells a fine-tuned and highly specific
response to luminal protein folding imbalance. However, when
the load of misfolded proteins increases they need to be retur-
ned to the cytoplasm to become proteasome substrates. This
clearance of malformed proteins from the ER is not a pas-
sive process, but uses the energy-dependent ER-associated
degradation pathway (ERAD). In sum, organisms harbor a variety
of protein quality control mechanisms to maintain proteome
integrity that, however, lose capacity with age, thereby increasing
the load of unfolded proteins and the susceptibility to proteo-
toxic diseases, such as Alzheimer’s or Parkinson’s. Consistent-
ly, improved protein homeostasis has been associated with
longevity in multiple species (Vilchez et al., 2014).
Recent work identified the hexosamine biosynthesis pathway
(HP) as a key player in both protein quality control and
longevity (Denzel et al., 2014). The first and rate-limiting enzyme
of the HP, glutamine-fructose-6-phosphate aminotransferase
(GFAT-1), shunts a fraction of fructose-6-phosphate from
glycolysis into the synthesis of UDP-N-acetylglucosamine
(UDP-GlcNAc). This activated aminosugar serves as a substrate
for a variety of glycosylation reactions including ER-specific
N-glycosylation, mucine-type O-glycosylation in the Golgi
apparatus, and addition of single O-GlcNAc moieties to Ser/
Thr residues. All of these posttranslational modifications play a
regulatory role in protein folding, Sekretion and function.
Moreover UDP-GlcNAc is a building block of highly abundant
biopolymers such as chitin and glycosaminoglycans. The
anabolic HP integrates various aspects of energy metabolism
and is therefore under tight control: The key enzyme GFAT-1 is
negatively regulated by the HP’s end product UDP-GlcNAc (Assrir
et al., 2014). In addition, the major energy-sensing AMP-activated
protein kinase (AMPK) as well as protein kinase A regulate
GFAT-1 activity through phosphorylation (Chang et al., 2000;
Hu et al., 2004).
Forward genetic screens in the nematode
Caenorhabditis
elegans
generated multiple GFAT-1 gain-of-function (gof)
alleles that increase the flux through the HP leading to
elevated levels of the product UDP-GlcNAc. Increased GFAT-1
activity not only overcomes toxicity caused by tunicamycin, a
drug inducing protein misfolding stress in the ER by blocking
N-glycan transfer, but also improves allover protein quality
control leading to longevity and protection from toxic protein
aggregation (Denzel et al., 2014). Importantly, supplementation
with the HP precursor N-acetylglucosamine (GlcNAc) recapitu-
RESEARCH NEWS
lates HP activation and, as seen in GFAT-1 gof mutants, improves
protein quality control. Surprisingly, increased flux through the
HP in GFAT-1 gof mutants does not induce canonical unfolded
protein response pathways, but activates protein degradative
processes including ERAD, proteasome activity, and autophagy,
all of which are required for the beneficial effects on an organism
level. The proteome-protective effects of HP activation in the
nematode also require N-glycosylation and specific branches
of the UPR
ER
, although both are not particularly elevated upon
GFAT-1 activation. It can be speculated that increased UDP-
GlcNAc levels in GFAT-1 gof mutants confer tunicamycin re-
sistance by directly competing with UDP-GlcNAc in glyco-
transfer reactions. However, it remains entirely elusive whether
the chaperone-independent improvement of proteome integri-
ty is mediated through enhanced overall glycosylation, glyco-
sylation of specific regulatory proteins, or also involves
glycosylation-independent components such as direct alloste-
ric functions of UDP-GlcNAc. Strikingly, increased HP flux does
not result in major changes of
C. elegans
transcriptional profile,
indicating that posttranscriptional events underpin the induction
of protein quality control mechanisms found in
C. elegans
GFAT-1 gof mutants.
In mammals, GFAT-1 is directly induced by one of the three
UPR
ER
branches. In particular, studies in cardiomyocytes demons-
trated that spliced X-box binding protein 1, a highly conserved
signal transducer of the UPR
ER
directly activates GFAT-1 expres-
sion (Wang et al., 2014). Ischemia/reperfusion in the heart was
shown to trigger XBP-1 splicing and the subsequent HP activa-
tion exerts robust cardioprotective effects. This protective stress
response is mediated via O-GlcNAcylation, which is in contrast
to findings in
C. elegans
, where key O-GlcNAcylating enzymes
are dispensable for lifespan extension by GFAT-1 gof. These con-
tradictory findings might be explained by the limited knowledge
about
C. elegans
O-GlcNAcylation networks, which leaves the
opportunity that there is remaining enzymatic activity even after
knockout of the key enzyme
ogt-1
.
Across species, increased activity of the HP via GFAT-1 activation
or GlcNAc supplementation results in downstream cytoprotec-
tive effects: In the nematode, this leads to enhanced systemic
fitness and longevity, while in mice it results in cardioprotection
through O-GlcNAcylation. Interestingly, supplementation with
the related amino sugar glucosamine (GlcN) results in lifespan
extension in both worms and mice, however these effects are
probably mediated via inhibition of glycolysis and subsequent
hormetic reactive oxygen species (ROS) signaling, independent
of the HP (Weimer et al., 2014). It is fascinating that small
metabolites such as GlcNAc, GlcN, and a variety of others
including trehalose or
α
-ketoglutarate can slow the aging process
throughdistinctmechanisms (Chin et al., 2014; Honda et al., 2010).
Thereby the HP pathway in particular might work as energy
sensor linking the metabolic state to proteome integrity and
survival. Despite this biological significance little is known about
the downstream mechanisms of increased HP flux. Thus, our
lab is actively investigating the role of the HP in mammalian
