BBA - General Subjects 1866 (2022) 130022

Contents lists available at ScienceDirect

BBA - General Subjects

journal homepage: www.elsevier.com/locate/bbagen

The role of humanin in natural stress tolerance: An underexplored

therapeutic avenue
Sanoji Wijenayake a, b, Kenneth B. Storey c, *
a
Department of Biology, Richardson College for the Environment and Science Complex, University of Winnipeg, Winnipeg, Manitoba, Canada
b
Department of Biological Sciences and the Center for Environmental Epigenetics and Development, University of Toronto, Toronto, Ontario, Canada
c
Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The discovery of humanin (HN/MTRNR2) 20 years ago blazed a trail to identifying mitochondrial
Humanin derived peptides with biological function.
Mitochondrial-derived peptides Scope: Humanin is associated with pro-survival, cytoprotective, anti-inflammatory, and anti-oxidative properties
Stress tolerance
and may play a role in reducing neurodegenerative and metabolic disease progression. Although the role of
Cytoprotection
Hibernation
humanin in vitro and in vivo laboratory models is well characterized, the regulation of humanin in natural models
Anoxia that encounter lethal cytotoxic and oxidative insults, as part of their natural history, require immediate research.
In this review, we discuss the conservation of humanin-homologues across champion hibernators, anoxia and
freeze-tolerant vertebrates and postulate on the putative roles of humanin in non-model species.
Significance: We hope characterization of humanin in animals that are naturally immune to cellular insults, that
are otherwise lethal for non-tolerant species, will elucidate key biomarkers and cytoprotective pathways with
therapeutic potential and help differentiate pro-survival mechanisms from cellular consequences of stress.

1. Introduction phenomenon that is documented in Monotremata (short-beaked

For most vertebrates, the best defence against harsh environmental
stress, including cold temperatures, prolonged starvation, dehydration,
and oxygen deprivation is to migrate to an environment with more
hospitable conditions or take shelter with sufficient food until conditions
return to normal. However, a select number of animals have adapted
natural stress tolerance strategies that allows them to transition into a
dormant-hypometabolic state, while remaining in their habitats. In
ectothermic vertebrates, such as amphibians, this dormant stage is
referred to as aestivation and it is a common hypometabolic strategy
utilized to survive arid summers [1], whereas freeze avoidance or freeze
tolerance is used to survive the cold winters [2]. Diapause is an adaptive
strategy that is often used by cold-hardy insects. Diapause temporarily
‘pause’ the developmental cycle to permit survival and/or to allow
hatching and maturation of the young during the breeding season when
resources are plentiful [2,3]. Select birds and endothermic mammals use
a hypometabolic strategy referred to as hibernation, that can range from
short bouts of night torpor to months of prolonged dormancy [4–6].
Hibernation is the best known and well characterized natural
echidna), Macroscelididae (elephant shrews), Rodentia (ground squir­
rels, marmots, prairie dogs, hamsters, and chipmunks), Microbiotheria
(monito del monte), Primates (lemurs), and Carnivora (little brown bats
and bears) [6], with the highest prevalence of obligatory hibernation
occurring in Rodentia and Carnivora. One of the adaptive values of hi­
bernation is to eliminate the need to maintain a constant, high body
temperature (Tb) during times of low energy production. During torpor,
Tb is maintained between 2 and 10 ◦ C in majority of temperate zone
hibernators, including Ictidomys tridecemlineatus (13-lined ground
squirrel), although a Tb of -3 ◦ C was reported in hibernating Spermophilus
parryii (Arctic ground squirrel) [4,5]. Moreover, in hibernating 13-lined
ground squirrels (13LGS), the heart rate reduces to 5 to 10 beats/min
from 350 to 400 beats/min, organ perfusion rate drops to <10% of
control conditions, and respiration drop to <1 breath/min from >40
breaths/min [5]. As such, most hibernators are at risk for ischemic
damage during entrance into torpor, experience extended exposure to
apnea during deep torpor, and variable rates of tissue perfusion and
reactive oxygen species (ROS) damage during bouts of arousal [6].
Consequently, they are an excellent natural model for the study of

* Corresponding author at: Canada Research Chair in Molecular Physiology, Institute of Biochemistry, Department of Biology, and Department of Chemistry, 1125
Colonel By Drive, Ottawa K1S 5B6, ON, Canada.
E-mail address: Kenneth_storey@carleton.ca (K.B. Storey).

https://doi.org/10.1016/j.bbagen.2021.130022
Received 30 June 2021; Received in revised form 19 September 2021; Accepted 4 October 2021
Available online 7 October 2021
0304-4165/© 2021 Elsevier B.V. All rights reserved.
S. Wijenayake and K.B. Storey
endogenous cytoprotective pathways and pro-survival mechanisms
against low temperature effects, starvation, and oxygen deprivation.
Hypoxia (low oxygen) and anoxia (complete lack of oxygen) arises
when the metabolic demand for oxygen exceeds the supply, and most
vertebrates, in particular humans, are extremely sensitive to oxygen
deprivation and reperfusion. Prolonged periods of hypoxia and anoxia
exposure can disrupt the regular functioning of so called “ATP sinks”,
including protein translation and degradation, urea biosynthesis,
gluconeogenesis, cell cycle, and various ion motive ATPases [7]. As
such, hypoxia and/or anoxia can rapidly lead to severe tissue and
neuronal damage and inevitable death to intolerant organisms. Among
vertebrates, long-term anoxia survival is highly developed in some
freshwater turtles, in particular red-eared sliders (Trachemys scripta
elegans), as well as subspecies of Chrysemys picta such as the Western
painted turtle (C. p. bellii), and Midland painted turtle (C. p. marginata).
Although humans cannot live without oxygen for more than a few mi­
nutes, sliders are able to survive underwater submergence without ox­
ygen for up to two weeks at 16 o C and for 12-18 weeks at 3 o C [8–10],
and return to normoxia without incurring cellular damage. Uniquely,
hatchlings of C. p. bellii and C. p. marginata are also able to survive whole
body freezing during their first winter which they spend in their
terrestrial nests; their high anoxia tolerance is crucial to their freeze
tolerance [2].
2. Biochemical mechanisms of natural stress tolerance
Hypometabolism or metabolic rate depression (MRD) is a key char­
acteristic of natural stress tolerance across endotherms and ectotherms.
Dramatic changes can be seen in the overall ATP turnover during
hypometabolism. For example, studies with isolated turtle hepatocytes
showed a 94% decrease in overall ATP turnover during long-term anoxia
[11], and 13LGS reduce their basal metabolic rate > 90% during hi­
bernation [5,12]. MRD is tightly coordinated through epigenetic
[12–15], transcriptional, post-transcriptional, and post-translational
modifications of cellular enzymes and signal transduction pathways
[4,12,16]. For example, multiple ATP-demanding cellular processes are
strongly suppressed, some very strongly (e.g. protein synthesis, protein
degradation, cell cycle, apoptosis, global gene expression, gluconeo­
genesis, and urea synthesis), and others less so (e.g. regulation of ion
motive ATPases) [9,10]. Robust tissue-specific increases in genes and
pathways involved in fuel reprioritization [17] (fatty acids in hiberna­
tors to glucose in anaerobic turtles), antioxidant defence, protein
chaperones, cytoprotective, and neuroprotective factors have been re­
ported during MRD [4,12] . Consequently, it is important to understand
that MRD does not translate to the shutting down of all biochemical/
cellular processes, rather it is the establishment of a ‘teeter-totter’ be­
tween ATP production and ATP utilization, where select cellular pro­
cesses are activated while others are repressed [12,18].
Hypometabolism is an excellent mode of natural stress tolerance,
however long-term viability of the organism depends largely on the
coordinated cell preservation/stress resistant strategies that are in place
during MRD and cytoprotective strategies that are utilized during exit
from MRD. In the case of 13LGS and most other mammalian hibernators,
during dormancy the emphasis is on the cell preservation and macro­
molecular repair mechanisms, where heat shock proteins (HSPs) [19],
glucose-regulated proteins (GRPs), and Nrf2 transcription factor [20],
among others, are upregulated. The arousal phase accompanies a
massive influx in oxygen consumption and tissue reperfusion, where in
ground squirrels, a 36-fold oxygen consumption is reported during
arousal [21]. This increase in oxygen consumption complements a
robust increase in ROS generation and lethal oxidative stress in both
intracellular and extracellular compartments. As such, significant spikes
in tissue-specific antioxidant responses were reported during arousal,
including plasma ascorbate [21], plasma melatonin [22], superoxide
dismutase (SOD) [23], catalase [24], glutathione peroxidase (GPox),
glutathione S-transferase (GST), peroxiredoxins, and metallothionein
2
BBA - General Subjects 1866 (2022) 130022
[5,6].
Similar cytoprotective mechanisms were reported in the champion
anaerobe, red-eared sliders. Hypometabolic turtles strongly suppress
most ATP consuming processes, while up-regulating selected genes and
proteins that serve cytoprotective functions, including transcription
factors (e.g. NFκB and Nrf2), antioxidant defenses (AO), HSPs), and the
unfolded protein response (UPR) [25–28]. The constitutively high
cytoprotective defenses seen in freshwater turtles during a low energy
anoxic-state is a result of natural adaptation, and/or preconditioning for
the rapid oxygen reperfusion that follows during the return to normoxia
[29]. The sudden reintroduction of oxygen during the recovery phase
leads to the overproduction of ROS and lethal levels of oxidative stress
across tissues and organs. Interestingly, turtles can tolerate severe
oxidative stress as they have well-developed and uncharacteristically
high levels of antioxidant defenses [28,29]. These include catalase, alkyl
hydroperoxide reductase, SOD (mitochondrial Mn-SOD and cytosolic
Cu/Zn-SOD), GPox, and GST [28–30].
Another fascinating aspect of hibernation and anoxia tolerance in
13LGS and red-eared sliders, respectively, is their neuronal resilience to
oxygen deprivation and oxidative damage. In ground squirrels, the ce­
rebral blood flow decreases to <10% during deep torpor, yet they fail to
sustain ischemia-induced neurological damage and do not suffer from
cognitive loss [31]. Instead, hibernating ground squirrels were shown to
regenerate complex synaptic structures, spines, and dendrites during
arousal [32]. Similarly, the brain of red-eared sliders exhibits minimal
neurodegeneration post months of continued anoxia and recovery [33].
Both these organisms employ extensive neuroprotective mechanisms to
prevent ATP shortages, irreversible collapse of ion gradients, and
oxidative damage in the brain, and thus are excellent animal models for
the identification of novel cytoprotective factors and biochemical
pathways [34] that can be used to develop therapeutics against human
neurological conditions, including strokes, aneurisms, umbilical cord
injuries, comas, and neurodegenerative disorders.
3. Humanin
The very first mitochondrial-derived peptide (MDP), humanin (HN/
MTRNR2), was discovered by the Nishimoto lab 20 years ago while
searching for novel anti-apoptotic proteins in the unaffected areas of the
brain of patients with Alzheimer's disease (AD) [35,36]. Two years later,
Guo et al., (2003) and Ikonen et al., (2003) reported that HN also exert
anti-apoptotic effects by binding exclusively to BAX in response to
cellular stress [37] and interacts with insulin-like growth factor-binding
protein 3 [38], respectively. HN is a small peptide (21-24 amino acids),
encoded within the 16S ribosomal RNA gene in the mitochondrial
genome (a gene-within-a-gene). HN was reported to protect cells and
organs against oxidative damage, apoptosis, heat shock, UV radiation,
chemotherapy, nutrient stress, toxic insults, as well as cardiac and ce­
rebral ischemia and hypoxia in vitro and laboratory in vivo models and
have been thoroughly reviewed elsewhere [39–44].
Since its discovery, 338 primary research articles have characterized
various biochemical and molecular biology (43%), neuroscience (30%),
cell biology (20%), endocrine and metabolic (17%), and pharmacolog­
ical (16%) properties of HN (Web of Science search, June/2021, with
“Humanin” as a topic and/or title). The mitochondrial form of Humanin
(M-HN) is highly conserved across many vertebrates ranging from pri­
mates to nematodes, suggesting an ancient evolutionary emergence
within the mitochondrial genome [39], however, pseudogenization of
the HN gene in the mitochondrial genome of vertebrates were reported
[45]. The term pseudogene was first coined by Jacq et al., (1977) to
describe multiple copies of genes found in the genome of the African
clawed frog (X. laevis) [46]. Majority of these duplicated gene copies
were non-functional yet retained by the genome. Pseudogenes are
typically formed through, 1) gene duplication events, and/or 2) muta­
tions in the CD (coding region) that causes the gene to stop producing a
functional protein due to the loss of the start codon, the introduction of a
S. Wijenayake and K.B. Storey BBA - General Subjects 1866 (2022) 130022

premature stop codon, frameshift mutations, and/or severe fragmenta­
tion of the exons [45]. Data mining of all the available human mito­
chondrial genomes in GenBank in 2017 by Logan (2017), discovered 17
HN variants of which 5 variants occurred at high frequency and were
associated with different mitochondrial haplogroups, where some lead
to changes in the amino acid sequence and others did not. 12 variants
occurred at low frequencies and the nature of these changes were
unverified in the study [45]. The study suggested that pseudogenization
of HN may not happen in humans, but a common feature in other ver­
tebrates. Through amino acid conservation testing across 16 vertebrates,
they identified Macaques (Macaca mulatta), the house mouse (Mus
musculus), cat (Felis catus), and the goat (Capra hircus) to have under­
gone pseudogenization, where the amino acid chain does not translate
into a functional peptide. Moreover, the author suggested that the
identification of HN pseudogenes in laboratory models, in particular
mice, to be a significant hinderance to interpreting a select number of
previously published studies addressing the therapeutic relevance of HN
in Alzheimer's disease. We disagree with this conclusion and postulate
that the previous use of mouse models to characterize the cytoprotective
effects of HN may not necessarily impede the therapeutic relevance of
the findings, rather the use of animal models that lack the functional
form of M-HN may hinder biological interpretations, due to the use of an
artificial system. There is little doubt synthetic and/or naturally trans­
lated M-HN provide cytoprotective benefits.
With this in mind, we conducted sequence prediction and
conservation as described in [47] to test for the presence of HN homo­
logues across vertebrates with natural stress tolerance (Fig. 1A, Table 1).
We also included three, non-hibernating rodent-models (house rat
(R. norvegicus), Eurasian red squirrel (S. vulgaris), and Swinhoe's striped
squirrel (T. swinhoei) that likely express functional HN homologues in
the analysis as controls. We found that hibernating tenrecs (E. telfairi),
marsupials (D. gliroides), and lemurs (M. murinus) express functional M-
HN isoforms with 65% sequence identity to HN (Fig. 1B), whereas hi­
bernating little brown bats (M. lucifugus) and David's myotis (M. davidii)
express a functional M-HN isoform with 74% sequence identity to HN
(Fig. 1C). Among hibernating rodents, Siberian hamster (E. sibiricus),
13LGS, and golden-mantle ground squirrel (C. lateralis) share 74%
sequence identity with HN and have functional M-forms (Fig. 1D).
Further, the champion anaerobes, red-eared sliders, and painted turtles,
express a functional M-HN isoform that share 56% sequence identity
with HN (Fig. 1E).
We also discovered the presence of pseudogenization among several
champion hibernators, anoxia, dehydration, and freeze-tolerant am­
phibians (Table 1). Naked mole rats (H. glaber), Richardson's ground
squirrels (U. richardsonii), black (C. ludovicianus) and white-tailed prairie
dogs (C. leucurus) do not express functional M-HN homologues at the
protein level, although they have highly conserved CDs at the genomic
level (Fig. 1F). Moreover, African clawed frogs (X. laevis) and tropical
clawed frogs (X. tropicalis), that are routinely included in laboratory
stress-studies, do not contain functional M-HN isoforms (Fig. 1G),

Fig. 1. Conservation of humanin and humanin-homologues across vertebrates. A) Mitochondrial coding region (CD), M and R isoforms of humanin, and 3D peptide
structure rendered graphically by ChimeraX molecular modelling using the NMR structure of humanin in 30% TFE (PDB ID: 1Y32), as illustrated originally by [47].
M-form denotes the peptide translated using the mitochondrial genetic code and R-form denotes the peptide translated from the cytoplasmic genetic code. B) Amino
acid conservation of humanin-homologues in hibernating tenrecs, marsupials, and lemurs. C) hibernating bats. D) hibernating rodents (house rat, Eurasian red
squirrel, and Swinhoe's striped squirrel is used in the analysis as non-hibernating controls). E) Rodents with natural stress tolerance lacking humanin. F) Anoxia-
tolerant freshwater turtles. G) Dehydration-tolerant amphibians lacking humanin. H) Freeze-tolerant amphibians lacking humanin. The predicted amino acid se­
quences are CDs translated using ExPaSy Bioinformatics Resource Portal. All multiple sequence alignments were done using Clustal Omega and rendered graphically
by EMBnet BOXSHADE 3.21 server. Black and “*” denotes conserved residues, gray denotes non-conserved amino acids but with similar categorization, and white
and “.” denotes non-conserved residues.

3
S. Wijenayake and K.B. Storey BBA - General Subjects 1866 (2022) 130022

perhaps due to gene duplication events [48]. Similarly, the freeze
tolerant wood frogs (R. sylvatica) and the gray tree frogs (H. versicolor)
lack a functional M-HN isoform (Fig. 1H), although gray tree frogs may
express a cytoplasmic isoform of HN (this remains to be experimentally
validated). The pseudogenization of the HN gene in select species with
natural stress tolerance could be related to their natural histories and
evolutional adaptations, where selective pressures to retain the func­
tionality of M-HN may have been less, compared to other species.
Moreover, these species employ alternate, well-coordinated mechanisms
of cytoprotection and anti-oxidation [4,12], such that HN may not be
required to fulfil the anti-apoptotic/pro-survival needs. It is important to
note that although many species appear to express functional HN iso­
forms, the true functionality of the peptide may differ to that of humans
and in vitro models [45]. As such, it is vital to combine in silico tech­
niques and experimental validation to understand the evolutionary
advantages of HN and its therapeutic potential. Also, it is imperative that
the true cytoprotective and anti-oxidative properties of HN should be
investigated in animal models that successfully tolerate lethal cellular
and redox insults as part of their life history and environmental adap­
tations to natural stress, as opposed to artificially inducing mild forms of
cellular stress in non-tolerant laboratory models. Majority of the time in
non-tolerant species, the biochemical pathways that are identified to be
upregulated are cellular consequences of the stress versus pro-survival/
cytoprotective mechanisms that are employed by the cells to fight
against the insult (a cellular reaction versus a cellular defence). The
inability to make this distinction in non-tolerant model systems can be
unfavourable to biopharmaceutical research aimed at developing HN-
related anti-neurodegenerative and anti-inflammatory therapeutics for
clinical use.

Table 1
Humanin (HN) homologues across mammals, reptiles, and amphibians. The predicted protein sequences are translated
using ExPaSy Bioinformatics Resource Portal with vertebrate mitochondrion (M-form) and standard (R-form) genetic
codes. Accession Number refers to the known and predicted humanin sequences as well as the complete mitochondrion
genomes on the NCBI databank. Open reading frames (ORFs) are highlighted in red.
Species Accession Amino Acid Sequence (5’-3’)
Number
H. sapiens AY029066*
M- Form MAPRGFSCLLLLTSEIDLPVK-RA-
(Human) R- Form MAPRGFSCLLLLTSEIDLPVKRRA-
ATG GCT CCA CGA GGG TTC AGC TGT CTC TTA CTT TTA ACC AGT GAA ATT GAC CTG
CCC GTG AAG AGG CGG GCA TAA
E. telfairi NC_002631.2# M- Form MATRGFNCLLLLISEIDLPVK-RGL
(Lesser hedgehog tenrec) R- Form MATRGFNCLLLLISEIDLPVKRRGL
ATG GCA ACA CGA GGA TTT AAC TGT CTC TTA CTT CTT ATC AGT GAA ATT GAC CTC
CCC GTG AAG AGG CGG GGA TTA
D. gliroides NC_005826.1# M- Form MAWRGFNCLLFSISEIDLPVQ-RGY
(Monito del monte) R- Form MA-RGFNCLLFSISEIDLPVQRRGY
ATG GCA TGA CGA GGG TTT AAC TGT CTC TTA TTC TCT ATC AGT GAA ATT GAC CTC
CCC GTG CAG AGG CGG GGA TAT
M. murinus NC_028718.1# M- Form MATRGFNCLLLLISEIDLPVK-REC
(Gray mouse lemur) R- Form MATRGFNCLLLLISEIDLPVKRREC
ATG GCT ACA CGA GGG TTC AAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TGT
M. davidii KM233172# M- Form MALRGFNCLLLSISEIDLPVK-RE-
(David’s myotis) R- Form MALRGFNCLLLSISEIDLPVKRRE-
ATG GCT CTA CGA GGG TTT AAC TGT CTC TTA CTT TCG ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGT GAA TAA
M. lucifugus KT901455.1# M- Form MAQRGFYCLLLLISEIDLPVK-RE-
(Little brown bat) R- Form MAQRGFYCLLLLISEIDLPVKRRE-
ATG GCT CAA CGA GGG TTT TAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGT GAA TAA
R. norvegicus KF011917# M- Form MAKRGFNCLLLSISEIDLPVK-LES
(House rat) R- Form MAKRGFNCLLLSISEIDLPVKRLES
ATG GCT AAA CGA GGG TTC AAC TGT CTC TTA CTT TCA ATC AGT GAA ATT GAC CTT
CCA GTG AAG AGG CTG GAA TCT
S. vulgaris AJ238588.1# M- Form MAPRGLNCLLLLISELAFPW-GGD
(Eurasian Red squirrel) R- Form MAPRGLNCLLLLISELAFP-RGGD
ATG GCC CCA CGA GGG TTA AAC TGT CTC TTA CTT TTA ATC AGT GAA CTT GCC TTC
CCG TGA AGA GGC GGG GAT AA
T. swinhoei KP027416# M- Form MAKRGFICLLLSISEIDLPVK-RE-
(Swinhoe’s stripped R- Form MAKRGFICLLLSISEIDLPVKRRE-
squirrel)
ATG GCT AAA CGA GGG TTT ATC TGT CTC TTA CTT TCA ATC AGT GAA ATT GAC CTC
CCC GTG AAG AGG CGG GAA TAA
E. sibiricus KF668525.1# M- Form MAWRGFNCLLLPISEIDLPVK-REY
(Siberian hamster) R- Form MA-RGFNCLLLPISEIDLPVKRREY

4
S. Wijenayake and K.B. Storey BBA - General Subjects 1866 (2022) 130022

ATG GCT TGA CGA GGG TTT AAC TGT CTC TTA CTC CCA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TAT
I. tridecemlineatus NC_027278.1# M- Form MAWRGFNCLLLLISEIDLPVK-REF
(13-lined ground squirrel) R- Form MA-RGFNCLLLLISEIDLPVKRREF
ATG GCT TGA CGA GGG TTT AAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TTT
C. lateralis KP698975.1# M- Form MAQRGFNCLLLLISEIDLPVK-REF
(Golden-mantle ground R- Form MAQRGFNCLLLLISEIDLPVKRREF
squirrel)
ATG GCT CAA CGA GGG TTT AAC TGT CTC TTA CTT TTG ATC AGT GAA ATT GAC CTC
CCC GTG AAG AGG CGG GAA TTT
H. glaber HQ689652.1$ M-Form MAKR-FFCLLLSVSEIDFPVK-RG-
(Naked mole rat) R- Form MAKRRFFCLLLSVSEIDFPVKRRG-
ATG GCC AAA CGA AGA TTT TTC TGT CTC TTA CTT TCA GTC AGT GAA ATT GAC TTC
CCC GTG AAG AGG CGG GGA TAA
U. richardsonii NC_031209.1$ M-Form MA-R-FDCLLLLISEIDLPVK-REF
(Richardson’s ground R- Form MA-RRFDCLLLLISEIDLPVKRREF
squirrel)
ATG GCT TAA CGA AGG TTT GAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TTT
C. ludovicianus NC_026706.1$ M-Form MAWR-FNCLLLLISEIDLPVK-REF
(Black-tailed prairie dog) R- Form MA-RRFNCLLLLISEIDLPVKRREF
ATG GCT TAA CGA AGG TTT GAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TTT
C. leucurus NC_026705.1$ M-Form MA-R-FNCLLLLISEIDLPVK-REF
(White-tailed prairie dog) R- Form MA-RRFNCLLLLISEIDLPVKRREF
ATG GCT TAA CGA AGG TTT AAC TGT CTC TTA CTT TTA ATC AGT GAA ATT GAC CTT
CCC GTG AAG AGG CGG GAA TTT
T.s. elegans NC_011573.1# M- Form MAKRGSTCLLQMISEIGLPVQKRG-
(Red-eared slider) R- Form MAKRGSTCLLQIISEIGLPVQKRG-
ATG GCC AAA CGA GGT TCT ACC TGT CTC TTA CAG ATA ATC AGT GAA ATT GGT CTC
CCC GTG CAA AAG CGA GGA TAA
Chrysemys picta belli NC_023890.1# M-Form MAKRGFNCLLLSISEIDLPVK-LES
(Painted Turtle) R- Form MAKRGFNCLLLSISEIDLPVKRLES
ATG GCT AAA CGA GGG TTC AAC TGT CTC TTA CTT TCA ATC AGT GAA ATT GAC CTT
CCA GTG AAG AGG CTG GAA TCT
X. laevis NC_001573.1$ M-Form PR-FNCLLHPIH-TDLRAEAGME
(African clawed frog) R- Form PRRFNCLLHPIH-TDLRAEAGIE
CCA CGA AGG TTC AAC TGT CTC CTG CAT CCA ATC CAT TAA ACT GAC CTC CGT GCA
GAG GCG GGG ATA GAA
X. tropicalis NC_006839.1$ M-Form MAPR-FNCLLHPIH-TDLPVQ-RGY
(Tropical clawed frog) R- Form MAPRRFNCLLHPIH-TDLPVQRRGY
ATG GCA CCA CGA AGG TTC AAC TGT CTC CTG CAT CCA ATC CAT TAA ACT GAC CTC
CCC GTG CAG AGG CGG GGA TAT
R. sylvatica NC_027236.1 M-Form TASRGLYCLLSPISETDLPVKKRGL
(Wood frog) R- Form TASRGLYCLLSPISETDLPVKKRGL
H. versicolor NC_045917.1 M-Form MAPR-LHCLLFLISKTNLPVKKRG-
(Gray tree frog) R- Form MAPRRLHCLLFLISKTNLPVKKRG-
ATG GCA CCA CGA AGG TTA CAC TGT CTC CTT TTT CTA ATC AGT AAA ACT AAT CTC
CCC GTG AAG AAG CGG GGA TAA
* Known humanin sequence.
#
Complete mitochondrial genome.
$
Species lacking the M and/or R-form of Humanin.

4. Humanin and natural stress tolerance
To date, HN has been experimentally validated in two natural model
systems that tolerate widespread cellular and redox insults. Szer­
eszewski and Storey (2019) identified a pro-survival and neuro­
protective peptide encoded within the 16S rRNA gene in the
mitochondrial genome of the obligate hibernator, 13LGS, and experi­
mentally characterized its regulation across six tissues and six stages of
torpor-arousal. According to the in-silico results, this unknown peptide
shares high structural (RMSD = 3.317 A) and sequence similarity (88%
conservation) to HN. They named this peptide, S-humanin [32]. More­
over, they reported elevated levels of S-humanin transcript and protein
in the cortex during late torpor, when compared to the euthermic con­
trol, and postulated that S-humanin may play a neuroprotective role in
the torpid brain. In particular, they explained that the attenuation of
neuronal damage, that is characteristic of hibernators, may be attributed
to the inhibition of BAX-mediated pro-apoptosis by S-humanin [37] in
the mitochondria during times of low energy. They also postulated that

5
S. Wijenayake and K.B. Storey
S-humanin may influence the increased expression of SOD [20] via the
ERK signaling pathway [49] and/or P13K and JAK/STAT pathways
[50], which are all shown to be upregulated in 13LGS by previous
studies.
In a subsequent study, using in silico protein techniques, including
sequence prediction/conservation, de novo (QUARK), and homology-
based protein modelling (iTASSER, SWISS-MODEL, and Phyre2), we
identified the presence of a novel HN-homologue in anoxia-tolerant red-
eared sliders, TSE-humanin [47]. We also found that M- form of TSE-
humanin share 74.7% and 56.2% sequence identity with HN at the
DNA and amino acid level, respectively. Like HN, TSE-humanin is
transcribed from a 75 bp region within the 16S rRNA gene in the
mitochondrial genome of T.s. elegans (a gene-within-a-gene) and the 3D
peptide structure of TSE-humanin as predicted by QUARK and iTASSER,
share 33-41% structural similarity within the core-alpha helix (amino
acid positions 3-19) with that of NMR-validated HN (PDB ID: 1Y32).
Moreover, we experimentally characterized the relative TSE-humanin
protein level across seven, anoxia-responsive tissues (brain, liver,
heart, kidney, spleen, red and while skeletal muscle) during control-
normoxia, 5 h anoxia, and 20 h anoxia. Interestingly, we found that
TSE-humanin protein concentration increase by >2-fold in the anoxic
turtle brain. This corroborates previous results that illustrates neuro and
cytoprotective properties of HN during cerebral ischemia and reperfu­
sion in non-tolerant species and in vitro models [51–54], albeit the in­
crease in HN levels were more prominent in the anaerobic sliders.
Further, the well characterized neuroprotective mechanisms that com­
bat ROS damage in the turtle brain, including HSPs, UPRs, antioxidants,
and anti-apoptotic factors [4,5] correspond to the increase in TSE-
humanin. We also found that TSE-humanin is robustly expressed in
anoxic-peripheral tissues, including liver, spleen, and skeletal muscle.
Especially in the liver, the increase in TSE-humanin could be associated
with insulin regulation, metabolic functions, and cytoprotection given
that anoxic-liver, the main storage site of glycogen, remain active and
proliferative during MRD [55] and utilize myriad of cytoprotective
strategies to limit oxidative damage.
We identified a curious amino acid complementarity between HN, S-
humanin, and TSE-humanin, where all three species share identical
amino acid sequences for positions 1-2, 4-5, 8-10, 14-16, and 18-20
(Fig. 2). This is important given previous studies have shown a signifi­
cant correlation between the placement of individual amino acids and
HN function. In particular, Cys8 (Cystein at position 8) that is critical for
BAX, BAD, and tBID binding and play a vital role in HN-mediated anti-
apoptosis [36] along with Leu9, Ser14, and Pro19 [44,56,57], are
conserved across the three species. Interestingly, these four amino acids
are also conserved across hibernating tenrecs, marsupials, lemurs, bats,
other hibernating rodents (Siberian hamsters, golden-mantle ground
squirrels), other anoxia-tolerant reptiles (painted turtles) (Fig. 1E), and
non-hibernating rodents such as house rat, red squirrel, and Swinhoe's
striped squirrel (Fig. 1F). Moreover, Leu9, Leu10, Pro19, and Val20 that
are crucial for HN secretion from tissues, are conserved across all above-
mentioned species, except for non-hibernating red-squirrels. Curiously,
red-eared slider is the only organism analyzed in this study, with natural
stress tolerance, that does not contain a Phe6, which is critical for
Insulin-like Growth Factor Protein 3 (IGFBP3) binding [38]. TSE-
humanin also do not contain the secondary site of IGFBP3 biding,
Lys21, although a closely related freshwater turtle, C.p.belli, express the
IGFBP3 binding sites. We postulate the lack of IGFBP3 binding sites in
TSE-humanin may be due to alternate cellular mechanisms that are in
place in red-eared sliders during anoxia that reduce the synthesis of
IGFBP3. In non-tolerant species, Hypoxia Inducible Factor-1-alpha (HIF-
1α) expression during extended hypoxia led to the activation of IGFBP3
and mitochondria-dependent apoptosis [58], however in red-eared
sliders HIF-1α activity is controlled by altering cyto/nuclear localiza­
tion as well as post-translational modifications of HIF-1α during
different stages of the anoxia-reperfusion cycle [59]. HIF-1α has shown
to be mostly active during the early hypoxia entrance and return to
6
BBA - General Subjects 1866 (2022) 130022
control levels during deep anoxia [16,60].
Another important amino acid that is conserved across 13LGS, red-
eared sliders, and humans is Ser14. Ser14 increase HN potency, isom­
erization, and protects neurons against AD-related genes and amyloid
beta aggregation [61]. Ser7 has also been shown to mediate similar ef­
fects, however the Ser7 substitution is not vital to HN-mediated neuro­
protection and is not conserved across S-humanin and/or TSE-humanin
(Fig. 2). Interestingly, neuronal mechanisms of hibernation and brain
anoxia tolerance are postulated to be inversely linked with the pathol­
ogy and cellular processes of neurodegenerative diseases and cognitive
impairments, including AD and dementia [32,47]. As such, neuro­
protective effects of humanin-homologues in natural model systems,
that can tolerate and survive lethal cytotoxic insults, should be charac­
terized to identify additional essential amino acids, and delineate novel
protein: protein interactions of HN.
5. Perspective
Since its discovery in 2001, HN and its analogues have shown
tremendous therapeutic potential against neurodegenerative (AD,
neuronal toxicity, learning, and memory impairments), metabolic
(glucose homeostasis, hyperlipidemia, diabetes mellitus, and obesity),
and cardiovascular conditions (atherosclerosis) and currently being
tested as an adjunct to chemotherapy. Although, the relationship be­
tween HN and tumorigenesis across different cancers and cancer
metastasis is inconclusive at present. Further characterization using
synthetic analogues, peptide mimics, and the use of gain and loss of
function experiments are needed to delineate the cytoprotective path­
ways, novel protein: protein interactions, tissue-specific regulation, and
long-term physiological roles of HN [57]. This is where the use of natural
model systems that regularly experience lethal levels of oxidative, pro-
apoptotic, and metabolic stress, as part of their natural histories and
evolutionary adaptations, is necessary. By using natural model systems,
researchers can differentiate defensive cytoprotective pathways that can
successfully combat lethal cellular stress (oxidative, ischemia/reperfu­
sion, low temperature, etc.) from cellular consequences of the exposure.
Nevertheless, the use of non-model systems to conduct therapeutic
research is not without limitation. The use of “model organisms”
including, laboratory rodents, yeast, nematodes, bacteriophages, and
fruit flies enable scientists to use a tractable and controlled system that
could be used to study a larger theme of biology, without incurring
molecular and biochemical features of the system itself [62]. As in, the
use of model systems is believed to remove confounding effects that are
associated with the natural history of the species and evolutionary
pressures that may have molded their physiology, biochemistry, and
behavioural responses. Moreover, the molecular tools and resources
(transgenic strains, species-specific antibodies, custom primers, estab­
lished baseline data, and deeply sequenced and annotated genomes) that
are readily available for model organisms surpass that of non-model
organisms. However, model organisms are not always the best systems
to study for all areas of biology. Especially human-centric therapeutic
and translational research that requires a high degree of repeatability
and an extensive understanding of the biochemical and molecular un­
derpinnings that are governed by species-specific natural histories and
evolutionary trajectories. Humans exhibit extensive genetic and epige­
netic variability and do not live in controlled laboratory environments
with minimal environmental pressures, and disease exposures. Although
the genome assembly of non-model species is lacking in depth and
annotation compared to laboratory models, the mitochondrial genomes
(Table 1) are sequenced and well annotated in most non-model species
with natural stress tolerance (available on National Center for Biotech­
nology Information (under Genome)). Therefore, in silico analysis of
MDPs and tests for pseudogenization can readily be conducted prior to
experimentally validating the presence and physiological functions of
MDPs in non-model species. Further, as the sequencing technologies
become more affordable and bioinformatic pipelines and resources are
S. Wijenayake and K.B. Storey
available for non-model species [63], it is now feasible to use non-model
organisms in translational research. Thus, we urge researchers to expand
animal models beyond that of in vitro and transgenic mice to understand
the cytoprotective effects of HN and other MDPs (MOTS-c and SHLPs).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the Natural Sciences and Engineering
Research Council (NSERC) of Canada. 1) Dr. Sanoji Wijenayake held a
NSERC postdoctoral research fellowship. 2) Dr. Kenneth B. Storey holds
a NSERC discovery grant (Funding #: 6793). In addition, Dr. Kenneth B.
Storey holds the Canada Research Chair in Molecular Physiology.
References
[1] A. Pinder, K. Storey, G. Ultsch, Aestivation and hibernation, in: M. Feder,
W. Burggren (Eds.), Enviromental Biol. Amphib, University of Chicago Press, 1992,
pp. 250–274.
[2] K. Storey, J. Storey, Natural freeze tolerance in ectothermic vertebrates, Annu. Rev.
Physiol. 54 (1992) 619–637, https://doi.org/10.1146/annurev.
ph.54.030192.003155.
[3] K.B. Storey, J.M. Storey, Insect cold hardiness: metabolic, gene, and protein
adaptation, Can. J. Zool. 90 (2012) 456–475, https://doi.org/10.1139/Z2012-011.
[4] K.B. Storey, J.M. Storey, Metabolic rate depression and biochemical adaptation in
anaerobiosis, hibernation and estivation, Q. Rev. Biol. 65 (1990) 145–174, https://
doi.org/10.1086/416717.
[5] K.B. Storey, Out cold: biochemical regulation of mammalian hibernation - a mini-
review, Gerontology 56 (2010) 220–230, https://doi.org/10.1159/000228829.
[6] H.V. Carey, M.T. Andrews, S.L. Martin, Mammalian hibernation: cellular and
molecular responses to depressed metabolism and low temperature, Physiol. Rev.
83 (2003) 1153–1181, https://doi.org/10.1152/physrev.00008.2003.
[7] P. Hochachka, P. Lutz, Mechanism, origin, and evolution of anoxia tolerance in
animals, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 130 (2001) 435–459,
https://doi.org/10.1016/s1096-4959(01)00408-0.
[8] G. Ultsch, D. Jackson, Long-term submergence at 3◦ C of the turtle, chrysemys picta
bellii, in normoxic and severely hypoxic water: survival, gas exchange, and acid-
base status, J. Exp. Biol. 96 (1982) 11–28.
[9] D. Jackson, Metabolic depression and oxygen depletion in the diving turtle,
J. Appl. Physiol. 24 (1968) 503–509.
[10] D.C. Jackson, Living without oxygen: lessons from the freshwater turtle, Comp.
Biochem. Physiol. Part A Mol. Integr. Physiol. 125 (2000) 299–315, https://doi.
org/10.1016/S1095-6433(00)00160-4.
[11] P. Hochachka, L. Buck, C. Doll, S. Land, Unifying theory of hypoxia tolerance:
molecular/metabolic defense and rescue mechanisms for surviving oxygen lack,
Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 9493–9498, https://doi.org/10.1073/
pnas.93.18.9493.
7
BBA - General Subjects 1866 (2022) 130022
Fig. 2. Amino acid sequence of M-humanin high­
lighting functionally relevant amino acids in human
(HN), the champion obligate hibernator 13-lined
ground squirrel (S-HN: Ictidomys tridecemlineatus)
and the anoxia tolerant red-eared slider turtle (TSE-
HN: Trachemys scripta elegans). Blue denotes amino
acids with positively charged side groups. Red de­
notes amino acids with negatively charged side
groups. Purple denotes amino acids with polar, un­
charged side groups. Green denotes amino acids with
nonpolar, aliphatic side group. ∆ = Pro-apoptotic
functions. Ω = IGFBP3 Binding site. Ò = Secretion. Æ
= Cell surface binding and cellular localization. ‡ =
Increase functional potency. The solid gray boxes
highlight amino acids that are conserved between HN
(humanin), S-HN (humanin homologue in 13-lined
ground squirrels) and TSE-HN (humanin homologue
in red-eared sliders). Solid blue boxes highlight amino
acids that are conserved between HN and S-HN. Solid
red boxes highlight amino acids that are conserved
between S-HN and TSE-HN.
[12] K.B. Storey, J.M. Storey, Metabolic rate depression in animals: transcriptional and
translational controls, Biol. Rev. Camb. Philos. Soc. 79 (2004) 207–233, https://
doi.org/10.1017/S1464793103006195.
[13] S. Wijenayake, L.J. Hawkins, K.B. Storey, Dynamic regulation of six histone H3
lysine (K) methyltransferases in response to prolonged anoxia exposure in a
freshwater turtle, Gene 649 (2018), https://doi.org/10.1016/j.gene.2018.01.086.
[14] S. Wijenayake, K. Storey, Dynamic regulation of histone H3 lysine (K) acetylation
and deacetylation during prolonged oxygen deprivation in a champion anaerobe,
Mol. Cell. Biochem. 1 (2020) 3, https://doi.org/10.1007/s11010-020-03848-x.
[15] S. Wijenayake, K. Storey, The role of DNA methylation during anoxia tolerance in a
freshwater turtle (Trachemys scripta elegans), J. Comp. Physiol. B. 186 (2016)
333–342, https://doi.org/10.1007/s00360-016-0960-x.
[16] K. Storey, J. Storey, Oxygen limitation and metabolic rate depression, in: K.
B. Storey (Ed.), Funct. Metab, John Wiley & Sons, Inc, Hoboken, NJ, USA, 2004,
pp. 415–442.
[17] S. Wijenayake, S.N. Tessier, K.B. Storey, Regulation of pyruvate dehydrogenase
(PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus), J. Therm.
Biol. 69 (2017) 199–205, https://doi.org/10.1016/j.jtherbio.2017.07.010.
[18] K.B. Storey, J.M. Storey, Biochemical adaptation to extreme environments, in:
Integr. Physiol. Proteomics Post-Genomics Age, 2005, pp. 169–200, https://doi.
org/10.1385/1-59259-925-7:169.
[19] H.V. Carey, N.S. Sills, D.A. Gorham, Stress proteins in mammalian hibernation,
Integr. Comp. Biol. 39 (1999) 825–835.
[20] M. Pier, Z. Ni, D.C. McMullen, K.B. Storey, Expression of Nrf2 and its downstream
gene targets in hibernating 13-lined ground squirrels, Spermophilus
tridecemlineatus, Mol. Cell. Biochem. 312 (2008) 121–129, https://doi.org/
10.1007/s11010-008-9727-3.
[21] H.M. Muleme, A.C. Walpole, J.F. Staples, Mitochondrial metabolism in
hibernation: metabolic suppression, temperature effects, and substrate preferences,
Physiol. Biochem. Zool. 79 (2006) 474–483, https://doi.org/10.1086/501053.
[22] D.X. Tan, L.C. Manchester, R.M. Sainz, J.C. Mayo, J. León, R.J. Reiter,
Physiological ischemia/reperfusion phenomena and their relation to endogenous
melatonin production: an hypothesis, Endocrine 27 (2005) 149–157, https://doi.
org/10.1385/ENDO:27:2:149.
[23] I. Okamoto, T. Kayano, T. Hanaya, S. Arai, M. Ikeda, M. Kurimoto, Up-regulation of
an extracellular superoxide dismutase-like activity in hibernating hamsters
subjected to oxidative stress in mid- to late arousal from torpor, Comp. Biochem.
Physiol. - C Toxicol. Pharmacol. 144 (2006) 47–56, https://doi.org/10.1016/j.
cbpc.2006.05.003.
[24] H. Ohta, I. Okamoto, T. Hanaya, S. Arai, T. Ohta, S. Fukuda, Enhanced antioxidant
defense due to extracellular catalase activity in Syrian hamster during arousal from
hibernation, Comp. Biochem. Physiol. - C Toxicol. Pharmacol. (2006) 484–491,
https://doi.org/10.1016/j.cbpc.2006.05.002.
[25] A. Krivoruchko, K. Storey, Molecular mechanisms of turtle anoxia tolerance: a role
for NF-κB, Gene 450 (2010) 63–69, https://doi.org/10.1016/j.gene.2009.10.005.
[26] A. Krivoruchko, K. Storey, Activation of the unfolded protein response during
anoxia exposure in the turtle Trachemys scripta elegans, Mol. Cell. Biochem. 374
(2013) 91–103, https://doi.org/10.1007/s11010-012-1508-3.
[27] A. Krivoruchko, K. Storey, Regulation of the heat shock response under anoxia in
the turtle, Trachemys scripta elegans, J. Comp. Physiol. B. 180 (2010) 403–414,
https://doi.org/10.1007/s00360-009-0414-9.
[28] A. Krivoruchko, K. Storey, Activation of antioxidant defenses in response to
freezing in freeze-tolerant painted turtle hatchlings, Biochim. Biophys. Acta - Gen.
Subj. 1800 (2010) 662–668, https://doi.org/10.1016/j.bbagen.2010.03.015.
[29] W. Willmore, K.B. Storey, Antioxidant systems and anoxia tolerance in a freshwater
turtle Trachemys scripta elegans, Mol. Cell. Biochem. 170 (1997) 177–185,
https://doi.org/10.1023/A:1006817806010.
S. Wijenayake and K.B. Storey
[30] M. Hermes-Lima, T. Zenteno-Savín, Animal response to drastic changes in oxygen
availability and physiological oxidative stress, Comp. Biochem. Physiol. Toxicol.
Pharmacol. 133 (2002) 537–556.
[31] S.N. Tessier, C.W. Wu, K.B. Storey, Molecular control of protein synthesis, glucose
metabolism, and apoptosis in the brain of hibernating thirteen-lined ground
squirrels, Biochem. Cell Biol. 97 (2019) 536–544, https://doi.org/10.1139/bcb-
2018-0256.
[32] K.E. Szereszewski, K.B. Storey, Identification of a prosurvival neuroprotective
mitochondrial peptide in a mammalian hibernator, Cell Biochem. Funct. 37 (2019)
494–503, https://doi.org/10.1002/cbf.3422.
[33] P.L. Lutz, S.L. Milton, Negotiating brain anoxia survival in the turtle, J. Exp. Biol.
207 (2004) 3141–3147, https://doi.org/10.1242/jeb.01056.
[34] Y.J. Lee, J.M. Hallenbeck, Insights into cytoprotection from ground squirrel
hibernation, a natural model of tolerance to profound brain oligaemia, in:
Biochem. Soc. Tran, 2006, pp. 1295–1298, https://doi.org/10.1042/BST0341295.
[35] Y. Hashimoto, Y. Ito, T. Niikura, Z. Shao, M. Hata, F. Oyama, I. Nishimoto,
Mechanisms of neuroprotection by a novel rescue factor humanin from swedish
mutant amyloid precursor protein, Biochem. Biophys. Res. Commun. 283 (2001)
460–468, https://doi.org/10.1006/bbrc.2001.4765.
[36] Y. Hashimoto, T. Niikura, H. Tajima, T. Yasukawa, H. Sudo, Y. Ito, Y. Kita,
M. Kawasumi, K. Kouyama, M. Doyu, G. Sobue, T. Koide, S. Tsuji, J. Lang,
K. Kurokawa, I. Nishimoto, A rescue factor abolishing neuronal cell death by a
wide spectrum of familial Alzheimer’s disease genes and Aβ, Proc. Natl. Acad. Sci.
U. S. A. 98 (2001) 6336–6341, https://doi.org/10.1073/pnas.101133498.
[37] B. Guo, D. Zhai, E. Cabezas, K. Welsh, S. Nouraini, A.C. Satterthwait, J.C. Reed,
Humanin peptide suppresses apoptosis by interfering with bax activation, Nature
423 (2003) 456–461, https://doi.org/10.1038/nature01627.
[38] M. Ikonen, B. Liu, Y. Hashimoto, L. Ma, K.W. Lee, T. Niikura, I. Nishimoto,
P. Cohen, Interaction between the Alzheimer’s survival peptide humanin and
insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis,
Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13042–13047, https://doi.org/10.1073/
pnas.2135111100.
[39] C. Lee, K. Yen, P. Cohen, Humanin: a harbinger of mitochondrial-derived peptides?
Trends Endocrinol. Metab. 24 (2013) 222–228, https://doi.org/10.1016/j.
tem.2013.01.005.
[40] Z. Gong, E. Tas, R. Muzumdar, Humanin and age-related diseases: a new link?
Front. Endocrinol. (Lausanne). 5 (2014) https://doi.org/10.3389/
fendo.2014.00210.
[41] T.L. Merry, A. Chan, J.S.T. Woodhead, J.C. Reynolds, H. Kumagai, S.J. Kim, C. Lee,
Mitochondrial-derived peptides in energy metabolism, Am. J. Physiol. -
Endocrinol. Metab. 319 (2020) E659–E666, https://doi.org/10.1152/
ajpendo.00249.2020.
[42] B. Miller, S.J. Kim, H. Kumagai, H.H. Mehta, W. Xiang, J. Liu, K. Yen, P. Cohen,
Peptides derived from small mitochondrial open reading frames: genomic,
biological, and therapeutic implications, Exp. Cell Res. 393 (2020), https://doi.
org/10.1016/j.yexcr.2020.112056.
[43] A. Rb Monteiro, G.C.G. Scarlato, D.F. Cavalini, G.E. Shiguemoto, Humanin, MOTS-
c and physical exercise: a new perspective, Biomed. Res. Rev. 3 (2019), https://doi.
org/10.15761/brr.1000129.
[44] C.F. Zuccato, A.S. Asad, A.J. Nicola Candia, M.F. Gottardo, M.A. Moreno Ayala, M.
S. Theas, A. Seilicovich, M. Candolfi, Mitochondrial-derived peptide humanin as
therapeutic target in cancer and degenerative diseases, Expert Opin. Ther. Targets
23 (2019) 117–126, https://doi.org/10.1080/14728222.2019.1559300.
[45] I.S. Logan, Pseudogenization of the humanin gene is common in the mitochondrial
DNA of many vertebrates, Zool. Res. 38 (2017) 198–202, https://doi.org/
10.24272/j.issn.2095-8137.2017.049.
[46] C. Jacq, J. Miller, G. Brownlee, A pseudogene structure in 5S DNA of Xenopus
laevis, Cell 12 (1977) 109–120, https://doi.org/10.1016/0092-8674(77)90189-1.
[47] S. Wijenayake, K.B. Storey, Oxidative damage? Not a problem!The characterization
of humanin-like mitochondrial peptide in anoxia tolerant freshwater turtles 40
(2021) 87–107, https://doi.org/10.1007/s10930-020-09944-7.
[48] T.L. Stark, D.A. Liberles, B.R. Holland, M.M. O’Reilly, Analysis of a mechanistic
markov model for gene duplicates evolving under subfunctionalization, BMC Evol.
Biol. 17 (2017) 1–16, https://doi.org/10.1186/s12862-016-0848-0.
BBA - General Subjects 1866 (2022) 130022
[49] S.N. Tessier, Y. Zhang, S. Wijenayake, K.B. Storey, MAP kinase signaling and Elk1
transcriptional activity in hibernating thirteen-lined ground squirrels, Biochim.
Biophys. Acta - Gen. Subj. 2017 (1861) 2811–2821, https://doi.org/10.1016/j.
bbagen.2017.07.026.
[50] S.M. Logan, S.N. Tessier, J. Tye, K.B. Storey, Response of the JAK-STAT pathway to
mammalian hibernation in 13-lined ground squirrel striated muscle, Mol. Cell.
Biochem. 414 (2016) 115–127, https://doi.org/10.1007/s11010-016-2665-6.
[51] X. Xu, C.C. Chua, J. Gao, R.C. Hamdy, B.H.L. Chua, Humanin is a novel
neuroprotective agent against stroke, Stroke 37 (2006) 2613–2619, https://doi.
org/10.1161/01.STR.0000242772.94277.1f.
[52] X. Xu, C.C. Chua, J. Gao, K.W. Chua, H. Wang, R.C. Hamdy, B.H.L. Chua,
Neuroprotective effect of humanin on cerebral ischemia/reperfusion injury is
mediated by a PI3K/Akt pathway, Brain Res. 1227 (2008) 12–18, https://doi.org/
10.1016/j.brainres.2008.06.018.
[53] S.T. Zhao, X.T. Huang, C. Zhang, Y. Ke, Humanin protects cortical neurons from
ischemia and reperfusion injury by the increased activity of superoxide dismutase,
Neurochem. Res. 37 (2012) 153–160, https://doi.org/10.1007/s11064-011-0593-
0.
[54] S.C. Zárate, M.E. Traetta, M.G. Codagnone, A. Seilicovich, A.G. Reinés, Humanin, a
mitochondrial-derived peptide released by astrocytes, prevents synapse loss in
hippocampal neurons, Front. Aging Neurosci. 11 (2019) 123, https://doi.org/
10.3389/fnagi.2019.00123.
[55] L. Buck, S. Land, P. Hochachka, Anoxia-tolerant hepatocytes: model system for
study of reversible metabolic suppression, Am. J. Phys. 265 (1993) 49–56, https://
doi.org/10.1152/ajpregu.1993.265.1.R49.
[56] A. Hazafa, A. Batool, S. Ahmad, M. Amjad, S.N. Chaudhry, J. Asad, H.F. Ghuman,
H.M. Khan, M. Naeem, U. Ghani, Humanin: a mitochondrial-derived peptide in the
treatment of apoptosis-related diseases, Life Sci. 264 (2021), 118679, https://doi.
org/10.1016/j.lfs.2020.118679.
[57] Z. Gong, E. Tas, R. Muzumdar, Humanin and age-related diseases: a new link?
Front. Endocrinol. (Lausanne). 5 (2014) 210, https://doi.org/10.3389/
fendo.2014.00210.
[58] C.C. Feng, C.C. Lin, Y.P. Lai, T.S. Chen, S. Marthandam Asokan, J.Y. Lin, K.H. Lin,
V.P. Viswanadha, W.W. Kuo, C.Y. Huang, Hypoxia suppresses myocardial survival
pathway through HIF-1α-IGFBP-3-dependent signaling and enhances
cardiomyocyte autophagic and apoptotic effects mainly via FoxO3a-induced BNIP3
expression, Growth Factors 34 (2016) 73–86, https://doi.org/10.1080/
08977194.2016.1191480.
[59] K. Biggar, A. Groom, K. Storey, Hypometabolism and turtles: physiological and
molecular strategies of anoxic survival, in: A. Nowakowska, M. Caputa (Eds.),
Hypometabolism Strateg. Surviv. Vertebr. Invertebr, Research Signpost, Kerala,
2011, pp. 57–94.
[60] K. Storey, J. Storey, Tribute to P. L., Lutz: putting life on `pause’ – molecular
regulation of hypometabolism, J. Exp. Biol. 210 (2007) 1700–1714, https://doi.
org/10.1242/jeb.02716.
[61] K. Terashita, Y. Hashimoto, T. Niikura, H. Tajima, Y. Yamagishi, M. Ishizaka,
M. Kawasumi, T. Chiba, K. Kanekura, M. Yamada, M. Nawa, Y. Kita, S. Aiso,
I. Nishimoto, Two serine residues distinctly regulate the rescue function of
humanin, an inhibiting factor of Alzheimer’s disease-related neurotoxicity:
functional potentiation by isomerization and dimerization, J. Neurochem. 85
(2003) 1521–1538, https://doi.org/10.1046/j.1471-4159.2003.01797.x.
[62] J.J. Russell, J.A. Theriot, P. Sood, W.F. Marshall, L.F. Landweber, L. Fritz-Laylin, J.
K. Polka, S. Oliferenko, T. Gerbich, A. Gladfelter, J. Umen, M. Bezanilla, M.
A. Lancaster, S. He, M.C. Gibson, B. Goldstein, E.M. Tanaka, C.-K. Hu, A. Brunet,
Non-model model organisms, BMC Biol. 15 (2017) 1–31, https://doi.org/10.1186/
S12915-017-0391-5.
[63] R.R. da Fonseca, A. Albrechtsen, G.E. Themudo, J. Ramos-Madrigal, J.A. Sibbesen,
L. Maretty, M.L. Zepeda-Mendoza, P.F. Campos, R. Heller, R.J. Pereira, Next-
generation biology: sequencing and data analysis approaches for non-model
organisms, Mar. Genomics 30 (2016) 3–13, https://doi.org/10.1016/J.
MARGEN.2016.04.012.