Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, Suppl. 1, pp. S36-S59 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Uspekhi Biologicheskoi Khimii, 2025, Vol. 65, pp. 55-90.

S36

REVIEW

Moonlighting Proteins of Human

and Some Other Eukaryotes. Evolutionary Aspects

Sergei S. Shishkin

Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences,

119071 Moscow, Russia

e-mail: sergeyshishkin@yandex.ru

Received February 7, 2024

Revised March 27, 2024

Accepted April 2, 2024

Abstract— This review presents materials on formation of the concept of moonlighting proteins and general

characteristics of different similar proteins. It is noted that the concept under consideration is based on the

data on the existence in different organisms of individual genes, protein products of which have not one,

but at least two fundamentally different functions, for example, depending on cellular or extracellular loca-

tion. An important feature of these proteins is that their functions can be switched. As a result, in different

cellular compartments or outside the cells, as well as under a number of other circumstances, one of the

possible functions can be carried out, and under other conditions, another. It is emphasized that the signif-

icant interest in moonlighting proteins is due to the fact that information is currently accumulating about

their involvement in many vital molecular processes (glycolysis, translation, transcription, replication, etc.).

Alternative hypotheses on the evolutionary origin of moonlighting proteins are discussed.

DOI: 10.1134/S0006297924602855

Keywords: moonlighting proteins, protein polymorphism, multiple protein functions, glycolytic enzymes,

ribosomal proteins, proteins of chromatin

INTRODUCTION

In modern biochemistry notions about proteins,

one of the main research objects of this science, are

developing constantly. In particular, already at the end

of XX century it was shown that expression of the

exon-intron genes of eukaryotes often occurs with al-

ternative splicing, and, as a result, one gene is capable

of synthesizing several sometimes very different pro-

tein products. It was found out later that these mac-

romolecules are subjected to individual or multiple

posttranslational modifications, which further expand

the set of products of functioning of a single gene.

Moreover, accumulated data indicate that in different

human individuals (and other eukaryotic organisms)

polypeptide chains determined by the same gene

could have slight differences, such as single amino

acid replacements due to the so-called point mutations

in this gene. All this knowledge and other available

data resulted in introduction of the notion of proteo-

forms to the scientific literature in 2013 [1].

The ability of single genes to produce a set of

proteoforms with certain structural and functional

features has been considered as a consequence of

continuing evolutionary processes facilitating acquir-

ing of many important traits by different organisms

such as, for example, resistance to environmental

stresses [2, 3].

At the same time, the data have been accumulat-

ed that proteoforms in general are involved in such

phenomenon as protein polymorphism, which is char-

acterized as existence in humans and other mammal

of several protein molecules with certain common fea-

tures, but with clearly pronounced differences, which

often are crucial for their functions [4]. Furthermore,

it has been suggested in numerous studies that the

existence of polymorphic proteins is the result of evo-

lution from the so-called last universal common an-

cestor, to mammals and humans, for example [5, 6].

Investigation of multifunctional proteins could be

considered as an important direction in the modern

studies of proteins. Almost three thousand of reviews

MOONLIGHTING PROTEINS OF HUMAN AND SOME OTHER EUKARYOTES S37

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig. 1. Graphic representation of bibliometric map created with the help of VOSreviewer program, which covers the subject

area limited to the words “multifunctional protein, review” and contains information on materials available in PubMed

database published during 2014-2024. Thematic clustering was carried out automatically based on the most often used key

words. In the graph center the node “moonlighting proteins” is highlighted.

on the features of such proteins exhibiting different

main functions (enzymatic, transport, regulatory, and

others) have been published in the recent decade,

which also have other ‘additional’ functions. These

publications have been annotated in the PubMed

database, which is for many years supported by

the National Center for Biotechnology Information

(NCBI) of the USA. General overview of these publi-

cation could be obtained with the help of bibliomet-

ric analysis performed with the help of the program

VOSreviewer.

VOSreviewer is a free computer program capable

of constructing graphic images of bibliometric maps of

certain subject areas [7-9]. In our case the subject area

was limited to the words ‘multifunctional protein, re-

view’. VOSreviewer provides visualization of the sub-

ject area based on analysis of the selected publica-

tions presented as a network between the sources in

a two-dimensional space. The formed network consists

of ‘nodes’ and ‘edges’ with nodes representing frequen-

cy of publications with certain key words and edges

existence of thematic associations between the nodes.

Graphic representation of a bibliometric map in-

cluding materials from PubMed database published in

the period from 2014 to 2024 is presented in Fig. 1.

Visualization was performed based on the most of-

ten used key words (around 100) using thematic clus-

tering.

As can be seen in Fig.1, there are 9 clusters (with

‘nodes’ marked in different colors). In the center of

the created map the node has been identified shown

as “moonlighting proteins”. This term was introduced

to the scientific literature more than twenty years ago

[10] and was used to describe a special group of mul-

tifunctional proteins.

In the second decade of XXI century these studies

have been advancing significantly. It has been noted

by many authors that some aspect of investigation

of “moonlighting proteins” are associated with the

fundamental principle of modern molecular biology

and emphasized that existence of such proteins corre-

sponds not to the formula “one gene– one function”,

but to the formula “one gene– two functions” [11-13].

Consequently, based on the abovementioned informa-

tion, the goal of this review was analysis of the results

of investigation of properties of “moonlighting pro-

teins” of human and of some other eukaryotes with

emphasis on evolutionary aspects.

EMERGENCE AND DEVELOPMENT

OF THE CONCEPT OF MOONLIGHTING

PROTEINS IN EUKARYOTES

Publications in the end of 1980s reporting exis-

tence in some organisms of certain genes with products

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

exhibiting not one, but at least two significantly differ-

ent functions manifested depending on, for example,

cellular or extracellular localization, could be con-

sidered as a prelude to formation of the concept of

“moonlighting proteins”. In particular, it was demon-

strated that the known cytoplasmic enzymes (lactate

dehydrogenase, enolase) are capable play a role of

structural crystallins in an eye lens [14, 15]. Accord-

ing to the presented data the indicated enzymes and

isoforms of crystallins have identical amino acid se-

quences.

Later Constance  J.  Jeffery  [10] in her review sum-

marized the available data on such proteins and sug-

gested a term “moonlighting proteins” to describe

these proteins. This unusual term originates from

an English slang word “moonlighting” referring to

a side job outside of normal working hours or to a

practice of theft cattle or crop destruction at night

times.

At present, increasing interest in moonlighting

proteins could be explained by the fact that numer-

ous publications are emerging on their involvement

in vital molecular processes (see, for example [16-18]).

However, a number of issues associated with moon-

lighting proteins remain unresolved during the recent

decades; in particular, it is poorly understood when,

how, and why such additional molecular functions

appeared. Although it has been suggested that this

property could evolve in the course of evolution [10,

19-21]. On the other hand, there is an alternative point

of view according to which the ancient proteins ini-

tially had several functions, which were gradually lost

during evolution leaving the protein with only one

function, enzymatic, for example [18].

One of the important characteristics of moonlight-

ing proteins is their ability for switching functions.

As a result, in different cellular compartments and

outside the cell, as well as under other specific con-

ditions one of the functions could be performed, and

under different conditions – another.

“Switching” of functions by moonlighting proteins

attract attention of researchers from the very begin-

ning of investigation of this phenomenon until now

(see, for example [10,  12,  22]). The mechanisms of

“switching” are actively examined, and among those,

the mechanisms associated with posttranslational

modifications have been considered [22,  23]. Among

the conditions causing “switching” of functions specif-

ic features of synthesis of moonlighting proteins in the

cells with different types of differentiation have been

considered, as well as changes in oligomeric composi-

tion of the protein (homo- or heterooligomers) associ-

ated or not associated with intracellular concentration

of a ligand, substrate, cofactor, or product, etc. [24, 18].

Many authors described also various combinations

of the variants of “switching” functions.

In this regard, it seems important to note that

starting from the end of the XX century and until now

the concept is being developed about the so-called in-

trinsically disordered proteins [25,  26]. Emergence of

this concept was facilitated by the success of genomic

projects. In particular, it has been established that the

major part of gene sequences encodes proteins that

do not have the ability for unassisted folding into a

globular structure [25]. These intrinsically disordered

proteins have long segments of amino acid sequenc-

es, which, likely, either unfold in solution, or form

non-globular structures with undetermined conforma-

tion. Moreover, the idea began to spread that under

physiological conditions such proteins (and/or protein

segments) do not have one unique three-dimensional

structure, but assume several interconverting confor-

mational states [26, 27].

One of interesting reflection of this concept with

regards to moonlighting proteins are the studies in-

dicating association between the ability of a single

polypeptide chain perform two (or more) significant-

ly different functions and the presence in such poly-

peptide chains of intrinsically disordered regions [27,

28-30]. It has been suggested that due to ability of the

intrinsically disordered regions in these proteins to

assume several interconverting conformational states,

they have the ability to realize different functions.

Ability of some moonlighting proteins to perform both

enzymatic and chaperon-like activity, as well as play

a role of an enzyme or a regulatory factor could be

mentioned as examples [20, 27, 29, 31].

Another important feature of moonlighting pro-

teins worth mentioning is that in the case of introduc-

tion of a point mutation to the coding gene, which dis-

rupt one of the functions of this protein, other function

could be preserved (see [18, 22, 32, 33] for examples).

Multifunctional proteins, which presumably

emerged as a result of fusion of ancestral genes, com-

monly are not considered as moonlighting proteins

[10, 34, 35]. Ability of these proteins to perform sever-

al functions usually is associated with the presence in

their polypeptide chain of special domains with each

of them performing only one function. Nevertheless, is

has been assumed that for performing different func-

tion different structural elements of polypeptide chain

of typical moonlighting proteins could be used.

The increasing interest in moonlighting proteins

and numerous unsolved issues related to them was,

likely, the reason for publishing at the end of the sec-

ond decade of XXI century of the special paper by

Constance J. Jeffery with a telling title “Protein moon-

lighting: What is it, and why is it important?” [20].

Inthis manuscript the author characterizes moonlight-

ing proteins as macromolecules capable of performing

“more than one physiologically relevant biochemical

or biophysical function within one polypeptide chain”.

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Fig.  2. Schematic representation of general knowledge on biosynthesis and distribution of moonlighting proteins in eu-

karyotic cells; arrows show distribution of moonlighting proteins in cellular compartments, where they perform the main

known function [10, 20, 23, 28, 42, 44].

This definition has been accepted by many research-

ers [12, 36].

However, it must be also mentioned that some

authors emphasize existence of certain problems in

using these criteria of moonlighting proteins, espe-

cially with regards to subunits of oligomeric protein

complexes (see, for example, [37, 38]). Moreover, some

authors suggest that if the protein contain several do-

mains, which are all required for performing different

functions, these proteins could be also considered as

moonlighting proteins [16].

In general, it is obvious that at present the concept

on moonlighting proteins has been formed, which is

manifested by thousands of publications that include

the term “moonlighting proteins” in their content and

annotated in the combined databases PubMed and

ScienceDirect. Vast majority of these studies are de-

voted to moonlighting proteins of eukaryotes (from

unicellular organisms to multicellular plants and ver-

tebrate animals). Around one third of all publications

are devoted to moonlighting proteins of bacteria and

significantly lower number of papers are devoted to

moonlighting proteins of archaea. Among the paper

on eukaryotic moonlighting proteins, majority, obvi-

ously, is on human moonlighting proteins.

It has been reported in a number of publications

that moonlighting proteins are present practically in

all cellular compartments and subcellular structures,

as well as in extracellular formations. Schematic rep-

resentation of the processes of biosynthesis and dis-

tribution of moonlighting proteins in eukaryotic cells

is shown in Fig. 2.

It has been established in the studies that inside

the cell individual moonlighting proteins are often

transported from one compartment to another, for

example, from cytoplasm to nucleus and back [39],

are integrated into mitochondria [40, 41] and cellular

membrane [23]. Moonlighting proteins in the extracel-

lular structures are transported there through secretion

from certain types of cells [42]. Moreover, some moon-

lighting proteins have been found in blood flow [43].

Biosynthesis of moonlighting proteins (same as

of all other cellular proteins) is realized via transla-

tion of respective mRNAs, after that the newly syn-

thesized polypeptide chains are subjected to various

posttranslational modifications and begin to perform

their main known functions and additional functions.

In many cases to begin performing one or another

function transport of the protein to various cellular

compartment is required such as transport into the

cell nucleus or integration into the cellular membrane

(Fig. 2).

Considering the abovementioned materials on

moonlighting proteins, it seems very interesting to

review the available information on eukaryotic pro-

teins involved in some metabolic processes and main-

tenance of some intracellular structures that emerged

in the early stages of evolution. Correspondingly,

data on moonlighting proteins among the glycolytic

enzymes, as well as moonlighting proteins partici-

pating in the processes of translation and those that

are components of chromatin and of cellular mem-

branes are presented in the following four sections

of this review.

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MOONLIGHTING PROTEINS

AMONG GLYCOLYTIC ENZYMES

It is known that glycolytic processes occur in the

cell cytoplasm and are observed in certain forms in all

investigated eukaryotes. Among the proteins charac-

terized as moonlighting, glycolytic enzymes were iden-

tified from the very beginning of investigation [10,

14, 15]. Similar glycolytic enzymes often carry out

their additional functions outside of the cell cytoplasm

in such locations as extracellular structures or cell nu-

clei. It is important to note that in the third decade of

XXI century new information on glycolytic enzymes,

which are considered moonlighting proteins, contains

to be published [45, 46].

Glycolysis is recognized as one of the most char-

acterized metabolic processes, which sometimes is

described as one of the central metabolic pathways

[47-49]. Phylogenetic approach to investigation of gly-

colytic enzymes revealed that this metabolic process

is indeed very ancient and existed in live organism

already at the time point when eukaryotes diverge

from prokaryotes, i.e. around 1800 million years ago

[47, 49]. Moreover, there is information that certain

reactions similar to glycolytic could occur prior to ad-

vent of living cells in prebiotic period [50].

After the first publications on enzymes partici-

pants of glycolysis (1930s-1940s) and until now gly-

colytic processes occurring in eukaryotes are tradi-

tionally described as a Embden–Meyerhof–Parnas

pathway (E-M-P). It has been established that as a

result of functioning of this pathway under aerobic

conditions glucose molecules are converted in the

course of ten consequent enzymatic reactions into

two pyruvate molecules. Under anaerobic conditions

these pyruvate molecules in the eleventh reaction

are reduced with the help of lactate dehydrogenase

to lactic acid. Detailed description of the reactions in

the E-M-P pathway could be found in numerous text-

books on biochemistry (see, for example[51]). Never-

theless, certain features and original schemes of re-

alization of the E-M-P pathway have been published

recently (see [18, 49, 52-54] for examples). One of such

schemes (with small modifications) is shown in Fig.3

for convenience of further discussion.

According to the accumulated data many glyco-

lytic enzymes have been found to be moonlighting

proteins (and in some organisms practically all, see

[21, 55] for examples). It is known that the human gly-

colytic enzymes and glycolytic enzymes of higher ver-

tebrates exhibit pronounced polymorphism (i.e., they

comprise groups of related proteins) [46, 47, 54, 55].

Moreover, structure of the same glycolytic en-

zymes in different organisms is quite conserved.

Atthe same the additional functions have been discov-

ered not for all isoforms; hence, the question remains

whether all similar isoforms should be considered to

be potential moonlighting proteins.

Numerous publications are available in the liter-

ature on polymorphism and multifunctionality of gly-

colytic enzymes [18, 21, 55]. That is why it seems in-

advisable to describe all glycolytic enzymes and their

isoforms with additional functions. Below information

on only three group of human glycolytic enzymes (full

names are shown in Fig. 3), among which there are

isoforms with established additional functions, are

considered as examples.

Hexokinases. It has been established that human

genome contains at least five genes encoding different

enzymes capable of catalyzing reaction of phosphory-

lation of hexose using ATP [56, 57]. Two of these genes

(HK1, HKDC1) are located in the segment 10q22.1, and

the rest– on other chromosomes: HK2– in the region

2p12, HK3 – in 5q35.2, and HK4 – in 7p13, according

Fig.  3. Generalized scheme of glycolytic breakdown of glucose (Embden–Meyerhof–Parnas pathway), according to [51-53].

Oppositely directed blue and red arrows indicate reversible reactions. Full names are shown for three types of enzymes,

which are moonlighting proteins; rest of the enzymes are shown using common designations. Additional explanations

are presented in the text.

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Table  1. General characteristics of human hexokinases and their various proteoforms, according to UniProt

and OMIM NCBI

Accepted names

and symbols of genes

Numbers

in UniProt/OMIM

Quantity of amino

acid residues (aa)

Manifestation of polymorphism

Natural variants*** PTM****

Hexokinase-1,

Brain form hexokinase), HK1

P19367/142600,

235700*

605285*

917** 577 3

Hexokinase-2,

Muscle form hexokinase, HK2

P52789/601125 917 797 3

Hexokinase-3, HK3 P52790/142570 923 965 1

Hexokinase-4,

glucokinase, HK4, GCK

P35557/138079,

125851*

125853*

465** 588

no data

available

Hexokinase domain-containing

protein 1, HKDC1

Q2TB90/617221 917** 916

no data

available

* Numbers in OMIM NCBI on pathological syndromes associated with mutations in the gene of this protein.

** Several isoforms are known differing in sizes of amino acid sequences:

P19367-1 – 917 aa, P19367-2 – 916 aa, P19367-3 – 921 aa, P19367-4 – 905 aa;

P35557-1 – 465 aa, P35557-2 – 466 aa, P35557-3 – 464 aa;

Q2TB90-1 – 917 aa, Q2TB90-2 – 806 aa, Q2TB90-3 – 736 aa.

*** Natural variants – number of proteoforms usually with single amino acid substitutions, according to UniProt.

**** PTM – number of possible posttranslational modifications of amino acid residues, according to UniProt.

to the data of the database ‘Online Mendelian Inheri-

tance in Man’ (OMIM, NCBI). Hence, it is obvious that

in humans there is a prominent multilocus polymor-

phisms of hexokinases.

Three of the five indicated genes (HK1, HK2,

HK3) provide synthesis of very similar protein prod-

ucts [58, 59]. They have molecular mass of ~100 kDa

and their amino acid sequences display 70% identity

[60]. These hexokinases contain two similar large do-

mains. The hexokinases 1 and 3 one of the domains

(C-terminal) is catalytic, and another (N-terminal) –

regulatory (see structures P19367, P52790 in UniProt

database) [58], while in the hexokinase 2 both do-

mains are catalytic (see structure P52789 in UniProt).

Protein products of two other genes (HK4 and HKDC1)

are significantly different in structural and functional

characteristics from the first three. At the same time,

expression of all cited hexokinase genes could result

in formation of numerous proteoforms through vari-

ous mechanisms. Furthermore, point mutations have

been discovered in some hexokinase genes that are

associated with certain inheritable diseases. Informa-

tion on these topics is presented in Table 1.

It has been established that in humans at least

protein products of the genes HK2 and HK4 have ad-

ditional functions, i.e. are moonlighting proteins [21,

61,  62]. In particular, it was demonstrated that these

enzymes play a role of ‘glucose sensors’ mediating glu-

cose-stimulated secretion of insulin in pancreatic cells.

Furthermore, protein products of the HK2 gene were

shown to have ability for interaction with mitochon-

drial membranes, as well as they exert cytoprotective

effect on the healthy and neoplastic cells [63]. In other

words, it was shown that additional functions of in-

dividual isoforms of hexokinases could play signifi-

cant role in the development of various pathological

processes.

Enolases. In the early 1970s information began

to emerge that several isoforms of enolases, enzymes

catalyzing conversion of 2-phosphoglyceric acid into

phosphoenolpyruvate, exist in different human tissues

(Fig.  3) (see [64] for an example). Later, the respec-

tive isoforms differing in their biochemical properties,

were designated as isoforms α, β, and γ. It has been

established in the process that α-enolase is present in

the cells with very different types of differentiation,

while β-enolase is specific for muscles, and γ-enolase–

for nerve cells [65,  66]. It is generally assumed that

in human cells enolase isoforms are usually present

as homodimers [54,  66,  67]. However, there are also

data on existence of heterodimers with composition

αβ and αγ.

Later, practically before the start of extensive

human genome investigations, three main genes en-

coding enolases were identified and mapped: ENO1

(1p36.23) – encoding α-subunit, ENO2 (12p13.31) –

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

encoding γ-subunit; ENO3 (17p13.2)– encoding β-sub-

unit (according to the OMIM NCBI database]. Inconsis-

tencies in designations of the two indicated genes and

their protein products (ENO2 – encoding γ-subunit

and ENO3 – encoding β-subunit) have developed his-

torically, but are still present. Existence of one more

gene in the human genome encoding enolase, ENO4,

is also known. This gene encodes the sperm-specific

isoform of enolase: A6NNW6 UniProt, 131375 OMIM

NCBI. The ENO4 gene is mapped to chromosome

10q25.3. The presented data indicate that multilocus

polymorphism of enolases exist in humans.

Thousands of publications are devoted to investi-

gation of human enolases. In particular, the PubMed

search using keywords ‘human enolase’ produced

around 10 thousand of publications, while search in

the ScienceDirect database – more than 20 thousand.

Based on this enormous amount of information sum-

marized in the UniProt database, it was revealed that

the products of expression of the ENO1 gene (P06733)

are present not only in cytosol, but also in cell nu-

clei, membranes, and outside the cell (blood plasma),

where they perform a number of functions in addition

to the catalytic one. Considering all accumulated data

many researchers characterize the indicated protein

products as moonlighting proteins that are capable

of binding DNA and regulate expression, play a role

of plasminogen receptor, and participate in a number

of pathological processes. Moreover, alpha-enolase is

designated as an oncomarker, and as a potential target

for chemotherapy of some malignant tumors [68].

Recently direct indication appeared that the pro-

tein products of the ENO2 gene could be considered

as moonlighting proteins [69]. These are no similar

data about the human proteins biosynthesis of which

is mediated by the ENO3 and ENO4 genes.

Lactate dehydrogenase. Human lactate dehydro-

genases (LDH) comprise a set of isoforms, which are

homo- or heterodimers formed by one or two sub-

units, respectively. These enzymes catalyzing the last

reaction of anaerobic glycolysis (Fig.  3) have been

investigated in great detail, their properties are de-

scribed in numerous biochemistry textbooks (see [51]

as an example), and detailed characteristics of the

subunits are presented in the UniProt database

(P00338; P07195; P07864). Brief information on these

polypeptide chains from the UniProt and OMIM NCBI

databases is presented below.

Three genes have been identified in the human

genome that encode LDH – LDHA, LDHB, and LDHC.

The LDHA gene is mapped to the 11p15.4 region. This

gene is expressed in many organs, but most active

expression is observed in skeletal muscles. Its protein

product is named subunit A or M (from the word

‘muscle’). The LDHB gene encodes subunit designated

as subunitB orH (from word ‘heart’), it is mapped to

the 12p12.2-p12.1 region. Expression of the LDHB gene

also has been observed in many organs, but sharply

dominates in the heart muscle. Moreover, it is known

that expression of the LDHC gene (synonym LDHX,

localization – 11p15.5-p15.3) is realized almost exclu-

sively in testes; and the corresponding subunit forms

homotetramer of the specific form of LDH.

Hence, functioning of polymorphic human LDHs

is characterized by the significant tissue-specificity,

which allows using isoforms of LDH as markers of

certain types of pathology, for example markers of ma-

lignant tumors [70, 71]. At the same time, a number

of researchers emphasized certain features of LDH iso-

forms, which provide basis for considering them not

as simple indicators of tumor presence, but consider

their involvement into activation of several oncogenic

pathways and into the process that make tumors inva-

sive. In other words, isoforms of these enzyme perform

functions not associated with enzymatic catalysis.

There are data indicating that the LDH isoforms,

products of the LDHA gene, are present in the cell

nuclei, where they bind DNA, interact with some DNA

polymerases, stimulate synthesis of DNA and DNA re-

pair after exposure to UV radiation [72, 73]. Moreover,

some authors directly associate protein products of the

LDHA gene as moonlighting proteins, which play func-

tions of a regulatory factors in the cell nucleus that

bind to transcription complexes [74]. It is important

to note that according to the data reported by Rose-

weir et al. [75], nuclear proteoforms determined by

the LDHA gene (isoform LDH-5) in the patients with

colorectal cancer are associated with poor prognosis.

Consequently, it could be assumed that significant con-

tribution to the tumor progression could be provided

by LDH isoforms serving as nuclear regulatory factors.

According to the UniProt data, protein products of

the LDHB and LDHC genes found in the cell cytoplasm

(P07195; P07864) so far have not been detected in the

cell nuclei. There are no indications in the PudMed

and ScienceDirect databases that the respective isoen-

zymes exhibit any additional functions. Hence, most

likely among all human LDH isoenzymes only protein

products of the LDHA gene are moonlighting proteins.

Concluding our consideration of the available in-

formation on glycolytic enzymes that are considered

as moonlighting proteins, it is important to note that

recently a detailed review has been published on this

topic, in which many other metabolic enzymes have

been characterised as moonlighting proteins [18].

MOONLIGHTING PROTEINS

PARTICIPATING IN TRANSLATION

Protein biosynthesis, one of the most import-

ant manifestation of life, which occurs as a result

MOONLIGHTING PROTEINS OF HUMAN AND SOME OTHER EUKARYOTES S43

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

of a complex of interconnected molecular processes,

quintessence of which is translation of genetic informa-

tion encoded in the nucleotide sequence of messenger

mRNAs into amino acid sequence of the correspond-

ing proteins [76]. Apparatus of translation includes the

most conserved cellular proteins and RNAs, which is

considered as the most reliable proof of common or-

igin of all forms of life [77,  78]. It has been assumed

at the same time that the processes of translation

emerged in the very early stages of evolution.

Various aspects of translation are being investi-

gated at present [79, 80]. It has been established that

translation in eukaryotes occurs not only cytoplasm,

but also in mitochondria, and in plants– also in chlo-

roplasts, where special protein-synthesizing systems

with ribosomes exist, which differ significantly from

cytoplasmic systems. Although in the third decade

of XXI century the protein-synthesizing systems of

mitochondria and chloroplasts still attract significant

attentions, information considered in this review will

be mostly about the studies of eukaryotic ribosomes

and translation factors functioning in composition

of cytoplasmic polyribosome complexes.

Ribosomal proteins. It is considered that ribo-

somes comprise the main of mass of polyribosomal

complexes, and about 50% of each ribosome compo-

sition include several tens of ribosomal proteins (RPs)

(see, for example, [76,  81,  82]). RPs are described as

individual relatively small polypeptide chains; the eu-

karyotic 80S ribosomes contain 79-80 types of RPs [83].

At present, after many decades of intensive in-

vestigation of RPs in organisms belonging to three

so-called main domains of life (prokaryotes, archaea,

eukaryotes) a number of important conclusions could

be made [79, 80, 82]. In particular, the accumulated

data on amino acid sequences and on a number of

other characteristics of RPs in bacteria, archaea, and

eukaryotes demonstrate high degree of homology be-

tween them. This provided the possibility to summa-

rize the available data and suggest an integral system

of RPs nomenclature, which was developed by the rep-

resentatives of 25 scientific organizations [82].

The suggested system stipulates that the homol-

ogous RPs are assigned the same name regardless of

the species. The name typically includes a capital let-

ter and a number (SX or LY), where the letters symbol-

ize belonging to the large(L) or small(S) subunit, and

numbers (X and Y) – integral numbers. In addition,

the names were supplemented with special prefixes.

Considering that the significant part of RPs was found

to be typical for all domains of life (prokaryotes, ar-

chaea, eukaryotes), these ribosomal proteins were

given the prefix ‘u’ (universal). Prefix ‘b’ was given

to those RPs that were found only in bacteria, and

prefix ‘e’ – to the RPs of eukaryotes and archaea. To

designate RPs of mitochondria and chloroplasts the

prefixes ‘m’ and ‘c’ were suggested, respectively (for

example, uL2m; uL2c).

The developed nomenclature suited to almost all

known at that time RPs (with rare exceptions); and

the summarizing table included 39 proteins of small

subunits and 61 proteins of large subunits [82].

However, this nomenclature has been used in

far from all publications, which creates certain prob-

lems for comparative analysis of the reported results,

because, for example, in the integral nomenclature

system there is a protein named uL16, which among

the human of yeast ribosomal proteins is sometimes

called L10 [82,  84].

It can be stated in conclusion that the suggested

classification reflects various information regarding

the facts that the ribosomal proteins belonging to the

evolutionary distant groups of organisms display pro-

nounced structural conservatism, which defines the

respective functional properties. Presence of structur-

al and functional conservatism among the majority

of ribosomal proteins allows suggesting that they ex-

isted already at the early stages of evolution [81, 85].

In the process of ribosome biogenesis in eukary-

otic cells dozens of RPs are first synthesized in cyto-

plasm followed by their transport into a nucleus and

next to nucleolus [79, 86-88]. Hence, before the start

of formation of ribosomal particles, RPs are in a free

state located, in particular, in cytoplasm.

In the next step, the eukaryotic ribosome bio-

genesis, which starts in a nucleolus, continues in a

cytoplasm, where immature ribosomal particles bind

certain free RPs [79,  86-88]. After that, the completely

formed ribosomes are incorporated into polyribosome

complexes and perform translation function.

In general, the accumulated data indicate pres-

ence of cytoplasmic pool of RPs. Investigation of free

cytoplasmic RPs and their functions during several

decades was carried out by the scientists from dif-

ferent countries (see [89-91] as examples). As a re-

sult, indications emerge that the individual RPs have

the so-called ribosomal and extra-ribosomal functions

[79,  92-94]. In particular, a table with information on

RPs with established extra-ribosomal function or with

presumed extra-ribosomal was presented in the re-

view by Wang et al. [94]. This table includes 21 RPs

of the small ribosomal subunit and 19 RPs of the large

ribosomal unit. It must be emphasized also that in the

case of different disruptions of ribosome biogenesis,

accumulation of free ribosomal proteins was observed

in the cells, which could also perform various extra-

ribosomal functions [95, 96].

The abovementioned information on RPs is sum-

marized and presented schematically in Fig. 4.

Among the extra-ribosomal function of RPs, fa-

cilitation of cell proliferation and differentiation, as

well as participation in the processes of apoptosis and

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig. 4. Schematic presentation of the processes of biosynthe-

sis and transport of ribosomal proteins in eukaryotic cells

(based on information reported in [86, 87]). Designations

as in Fig.2.

DNA repair have been mentioned [94]. It has been

also mentioned that some of ribosomal proteins per-

form their extra-ribosomal functions through interac-

tion with certain cytoplasmic and/or nuclear proteins

[90, 97]. Furthermore, the ribosome-free state of some

ribosomal proteins allows their post-synthetic modifi-

cations (such as uS5, for example) and perform var-

ious extra-ribosomal functions including activation

of p53-dependent or p53-independent pathways as a

response to stress. In addition, it has been reported

that the ribosomal protein termed L13a (uL13, accord-

ing to the integral nomenclature) has extra-ribosomal

functions in the breast cancer cells [98]. Elucidation

of the role of free ribosomal proteins (i.e. their extra-

ribosomal functions) in the processes of oncogenesis

of various types of tumors could be considered as a

key result of such studies [99].

A number of publications of the Russian scien-

tists have been devoted to extra-ribosomal functions

of certain ribosomal proteins [100-102]. It was noted

in these works that the eukaryotic protein S3 partici-

pates in DNA repair, as well as in selective regulation

of gene activity, induction of apoptosis, and other mo-

lecular processes. In addition, the eukaryotic protein

S15 (uS19, according to the integral nomenclature)

plays a function outside of the ribosome associated

with participation in regulation of tumor suppressor.

Moreover, it has been emphasized that mutations

in the gene encoding the S15 protein are associated

with the development of certain diseases (Diamond–

Blackfan anemia, chronic lymphocytic leukemia, Par-

kinson’s disease) [101]. And finally, in the recently

published paper Ochkasova et  al.  [102] considered

possible extra-ribosomal functions of RPs in eukary-

otes associated with their participation in cell-cell

communications via extracellular vesicles including

exosomes.

It could be concluded based on the available data

that many RPs have established extra-ribosomal func-

tions, and, hence, could be considered as moonlighting

proteins (see, for example [79, 96, 103, 104]). Research

on the extra-ribosomal activities of RPs is continuing,

and recently Mołoń et al. [105] demonstrated that

the uL6a plays a key role in the cell response to ox-

idative and osmotic stress, and could be considered

as a moonlighting protein.

Translation factors. It is known that several

groups of protein factors participate in functioning

of polyribosome complexes, which are required for

mediating certain steps of translation (initiation, elon-

gation, and termination). Among those, moonlighting

proteins have been identified in different organisms

[106,  107]. It has been recognized that at least 12

proteins termed eukaryotic initiation factors(eIF) are

responsible for translation initiation facilitating in-

teraction of the small ribosomal subunit (40S) with

5′-untranslated regions (UTR) in mRNA. The canonic

cap-dependent translation initiation involved binding

of eIF4F with 7-methylguanosine cap (m7G cap) at

the 5′-end of mRNA, and eIF4G1 plays a central role

acting as multipurpose ribosome adapter connecting

other eIFs, such as eIF4E, eIF4A into the specialized

initiation complexes [106, 107].

Since the first decade of XXI century, publica-

tions started to emerge reporting that some eIFs are

present in nuclei, where they perform additional

functions associated with regulation of transcription,

processing, and mRNA export (see, for example, [106,

108]). In the process it was found out that some eIFs

interacting with the 5′-cap of mRNA and regulating

global translation have additional functions involving

their ability to bind certain proteins outside of polyr-

ibosome complexes [109]. It is worth mentioning that

there is information that eIF5A, which is considered

to be highly conserved in eukaryotes and archaea,

also exhibits endoribonuclease activity [110].

Among numerous studies devoted to investi-

gation of eukaryotic translation elongation factors

(eEF), there a number of studies indicating that these

proteins have additional functions (see, for exam-

ple, [111-113]). In particular, more than 20 years ago

EjiriS. presented information (2002) [111] in the large

review on the role of eEF-1 subunits not only in medi-

ating translation in polyribosome complexes, but also

on the ability of these proteins to bind actin, as well

as ability to interact in the case of nuclear localization

with the zinc-finger protein R1.

It has been demonstrated recently that the eu-

karyotic translation elongation factor eEF1A2 has cell

membrane and organelle membrane binding sites,

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

and, moreover, its involvement in some molecular

processes has been shown [114]. As a result, the cit-

ed authors noted that considering new information

on the role of eEF1A2 in autophagy, oncogenesis, and

virus replication, modern notions on significance of

this ancient protein go far beyond its canonical func-

tion involving delivery of aminoacyl-tRNA to ribosome.

It seems important to mention the recent review

by Negrutskii et al. [113], which summarizes current

information on the family of mammalian translation

elongation factors eEF1 obtained as a result of many

years of work by several generations of scientists.

Negrutskii  et  al.  [113] analyzed the data on spatial

organization and posttranslational modification of

eEF1A1 and eEF1A2, as well as provided examples of

their participation in the processes not associated with

translation. It was emphasized that although both

variants of eEF1A exhibit similar activity in transla-

tion, they could differ with regard their additional

functions.

According to the opinion of a number of authors,

termination is a crucial step of translation; in particu-

lar, it is important that the premature termination of

translation could result in generation of toxic truncat-

ed peptides (see, for example [115]). Three proteins

termed release factors and designated as RF1, RF2,

and RF3 mediate translation termination. According

to the data reported by Chai et al. [116] the eukary-

otic release factor RF3 (eRF3) is present both in cyto-

plasm and in the nucleus, where it presumably par-

ticipates in morphological organisation of the nucleus.

Correspondingly, these authors suggested to consid-

er eRF3 as a polyfunctional protein with additional

roles in the process of protein synthesis termination.

Later it was demonstrated that eRF3 indeed is a poly-

functional protein that plays a key role in translation

termination, and also participates in initiation of

mRNA decay and regulation of apoptosis [117].

Hence, there are convincing data indicating that

many proteins involved in the processes of translation

(which are realized in cytoplasm) are present also in

the cell nuclei, where they play various additional

functions.

CHROMATIN PROTEINS

WITH ADDITIONAL FUNCTIONS

Nuclei of eukaryotic cells are described as large

organelles with majority of the space occupied by

chromatin containing genomic DNA, histone and

non-histone proteins, as well as various types of RNAs

[118, 119]. Chromatin is well-known as a rather dy-

namic formation containing different structural ele-

ments (termed ‘regions’, ‘compartments’, ‘territories’,

and others, see examples in [119]). It is commonly

recognized that chromatin has two main regions:

euchromatin and heterochromatin. In general, eu-

chromatin is defined as less dense genome region

enriched with transcriptionally active genes, and het-

erochromatin – as a region consisting of more dense

genome regions containing transcriptionally inactive

genes [120, 121]. Among the multiple components of

chromatin, proteins have been identified that have,

in addition to nuclear, other localizations, which in

addition to the function of maintaining chromatin

structure were shown to have additional functions.

In other words, some chromatin proteins are char-

acterized as multifunctional, and some are directly

assigned to moonlighting proteins. It is assumed that

clear polymorphism of chromatin proteins is a result

of directed evolution [122-124].

Histones. The main histones in vertebrates, in-

cluding humans (H1, H2A, H2B, H3, and H4), are the

most well-investigated eukaryotic proteins, which

provide basis for chromatin formation via specific

interactions with genomic DNA [125-127]. In these in-

teractions four pairs of histones H2A, H2B, H3, and

H4, which sometimes are called core histones, form

special octameric complexes – nucleosomes. Existing

estimates show that DNA molecule wraps around each

nucleosome (1.67 turn, 147 bp), and histone H1 pre-

sumably plays a role of an internucleosomal linker.

Histones are assigned to ancient proteins, precur-

sors of which initially emerged, as suggested, in the

so-called last universal common ancestor [126, 128].

Later, symbiosis of ancient bacteria with archaea re-

sulted in emergence of eukaryotic cells, in which for-

mation of the set of histones continued; and that pro-

vided a possibility of evolution progressing towards

increase of genome size [124, 126].

It is known that the exon-intron structure is a

specific feature of eukaryotic protein-coding genes,

while the similar genes in prokaryotes to not contain

introns. However, the intronless genes exist also in

higher eukaryotes. In particular, existing estimates

show that 3% of the genes in human genome are in-

tronless, and among those 20% are genes encoding

histones [122]. In other words, it could be hypothe-

sized that the histone genes in eukaryotes that have

come a long way in evolution, preserve some features

of the genes-analogues in prokaryotes.

Comparison of the yeast and human genomes

could provide some information on certain features of

evolution of histone genes on the path from single-cell

eukaryotes to multicellular organisms.

It has been reported [129] that the genes encod-

ing histones H2A, H2B, H3, H4 in the Saccharomy-

ces cerevisiae genome, are organized into four loci

(HTA1-HTB1; HTA2-HTB2; HHT1-HHF1; HHT2-HHF2)

each of them on containing two histone genes di-

vergently transcribed from the central promoter.

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Table 2. General characteristics of the human histone1 family, their genes, and polymorphism manifestations

according to [130] as well as UniProt and OMIM NCBI databases

Histone designations*

(names and synonyms),

symbols of genes

Numbers

in UniProt/OMIM

(chromosome

localization)

Quantity

of amino acid

residues

Natural

variants***

Number

of amino acid

residues

with PMTs****

H1.0 [Histone H1.0, Histone H1′,

Histone H1(0)] H1-0, H1F0, H1FV

P07305**, 142708

(22q13.1)

194 156 6

H1.1 [Histone H1.1, Histone H1a]

H1-1, H1F1, HIST1H1A

Q02539, 142709

(6p22.2)

215 352 >20

H1.2 [Histone H1.2, Histone H1c,

Histone H1d, Histone H1s-1] H1-2,

H1F2, HIST1H1C

P16403, 142710

(6p22.2)

213 596 >50

H1.3 [Histone H1.3, Histone H1c,

Histone H1s-2] H1-3, H1F3, HIST1H1D

P16402, 142210

(6p22.2)

221 70 >25

H1.4 [Histone H1.4, Histone H1b

Histone H1s-4] H1-4, H1F4, HIST1H1E

P10412, 142220

(6p22.2)

219 123 >30

H1.5 [Histone H1.5, Histone H1a

Histone H1b, Histone H1s-3] H1-5, H1F5,

HIST1H1B

P16401, 142711

(6p22.2)

226 410 >30

H1.6 [Histone H1t, Testicular H1 histone]

H1-6, H1FT, H1T, HIST1H1T

P22492, 142712

(6p22.2)

207 47 >10

H1.7 [Histone H1.7, Testis-specific H1 histone,

Haploid germ cell-specific nuclear protein 1,

Histone H1t2] H1-7, H1FNT, HANP1

Q75WM6, 618565

(12q13.11)

255 305 1

H1.8 [Histone H1.8, Histone H1oo, osH1]

H1-8, H1FOO,

H1OO, OSH1

Q8IZA3*****,

142709 (6p22.2)

346 321 no data

H1.10 [Histone H1.10, Histone H1x]

H1-10, H1FX

Q92522, 602785

(3q21.3)

213 189 >10

* Histone designation according to the new standardized nomenclature [130].

** There are reasons to suggest the possibility of formation of two isoforms P07305-1 and P07305-2 due to posttranslational

modification of pre-mRNA of histone H1.0.

*** PTM – number of possible posttranslational modifications of amino acid residues, according to UniProt.

**** Natural variants – number of proteoforms with, as a rule, single amino acid substitutions according to UniProt.

***** According to UniProt data Q8IZA3, there are two isoforms, differing in the size of amino acid sequences: Q8IZA3-1 –

346 aa, Q8IZA3-2 – 207aa.

It was found out that two genes determining biosyn-

thesis of histone H2A differ slightly from each other;

as a result, two isoforms of this protein are formed

in Saccharomyces cerevisiae. The same situation was

observed for the histone H2B. However, the loci HHT1-

HHF1 and HHT2-HHF2 contain each a pair of identical

genes, encoding identical proteins H3 and H4. In ad-

dition, three more histone genes have been identified

encoding proteins H1 (HHO1), H2AZ (variant close to

H2A), and a special centromeric H3 (CSE4). In total,

11 histone genes were found in the Saccharomyces

cerevisiae gene [129].

It is important to note that according to the infor-

mation presented in UniProt database, some histones

in yeasts in addition to the main structural functions

have additional functions associated with repair of

DNA damages (such as P04911 UniProt) or regulation

of transcription (Q12692 UniProt).

Unlike in the yeast genome, more than 70 his-

tone genes and a number of pseudogenes were found

in the human genome [127, 130]. It was shown that

these genes also form four clusters and encode pro-

teins belonging to five respective families (H1, H2A,

H2B, H3, H4) [127, 130, 131]. The largest gene cluster

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

is located on the chromosome 6 (locus HIST1) and

three more clusters (loci HIST2, HIST3, and HIST4) –

on other chromosomes (according to the OMIM NCBI

database). It was found out that the histone genes

and encoded proteins comprising respective fami-

lies are similar in many respects, but far from being

identical (for example, according to UniProt database,

Table 2).

Moreover, it has been noted that the histone H1.8

is localized both in the cell nucleus and in cytoplasm.

It must be mentioned that different classifications

and designations of individual histone genes and their

products, histones, exist in the literature. To avoid

problems and inconsistencies emerging due to this,

several attempts for unification of the used designa-

tions have been made; and recently a new standard

nomenclature of mouse and human histones has been

suggested [130]. This nomenclature including data on

such genes in humans and mice was created with

participation of the Human Genome Organization No-

menclature Committee (HGNC). Nevertheless, in many

sources including UniProt and OMIM NCBI databas-

es variety of the names of histone families are still

in use, which must be taken in consideration during

analysis of the literature data. General characteristics

of the human Histone1 family, their genes, and man-

ifestations of polymorphisms are presented above in

Table 2 as an example.

It follows from the data presented in the UniProt

database that all representatives of the H1 histone

family play similar functions in the chromatin struc-

ture, binding DNA sites located between nucleosomes,

and, thus facilitate formation of a macromolecular

structure known as chromatin fiber. In addition, the

functions of regulators of transcription of specific

genes, which are associated with chromatin remod-

elling and DNA methylation, have been assigned to

some H1 histones (see examples of P16403, P16402 in

UniProt).

Interestingly enough, some of the H1 histones

were suggested to be involved in splicing functions

[132, 133]. In particular, the histone H1.5 has the abili-

ty to bind DNA at the sites of splicing of short exons in

human lung fibroblasts. It was concluded, as a result

of investigation, that H1.5 participates in regulation

of selection of the splicing site and alternative splic-

ing, i.e., it has additional function [132]. Recently it

was shown in the model experiments that the histone

H1.2 exhibits high affinity to exons, while H1.3 binds

to intron sequences [133]. As a result, due to their

effect on elongation performed by RNA-polymerase  II,

conditions are created for exon skipping and/or intron

retention.

It seems important to emphasize that while the

representatives of human H1 histone family have

similar general functions, they differ significantly in

thesize of amino acid sequences (Table2); and some

of their genes display certain specificity of expres-

sion. According to the data from UniProt database,

expression of the majority of the genes of H1 histone

family is accompanied by appearance of hundreds of

proteoforms that have, as a rule, single amino acid

substitutions, as well as various posttranslational

modifications of amino acid residues. Corresponding-

ly, it can be assumed that, firstly, numerous isoforms

and variants of H1 histones are the products of long

evolution, and, secondly, a number of those perform

not only structural functions in chromatin composi-

tion, but also have additional functions.

Situation with the human core histones is much

more complicated that with the H1 histones. A whole

spectrum of the genes that determine biosynthesis of

a large sets of isoforms and variants of histones H2A,

H2B, H3, H4 were found in the human genome. These

data continued to be detailed. In particular, informa-

tion on 17 human genes encoding H2A histones has

been reported in 2020 [134], while more than 26 of

such gene have been identified in the 2022 study (al-

though, some of them have been noted as ‘variants

of histone H1 or H3’) [130]. Description of functions

of many human core histones in the UniProt data-

base, such as, for example, description of the histone

H2A type 2-B (Q8IUE6 UniProt) includes the following:

“Core component of nucleosome. Nucleosomes wrap

and compact DNA into chromatin, limiting DNA acces-

sibility to the cellular machineries, which require DNA

as a template. Histones thereby play a central role in

transcription regulation, DNA repair, DNA replication

and chromosomal stability. DNA accessibility is regu-

lated via a complex set of post-translational modifica-

tions of histones, also called histone code…” In other

words, it is emphasized that these proteins have, in

addition to the main structural function, a number of

additional functions. It seems reasonable to mention

that thousands of publications have been devoted only

to the function, which is termed ‘histone code’ (see,

for example [135]).

It is known that histones are synthesized in cy-

toplasm and, next, are transported through nucleo-

lemma to nucleus [136]. Nuclear import of histones

is realized with the help of proteins from the family of

karyopherin nuclear transport receptors, also known

as importins. Hence, after synthesis histones remain

for some time outside of chromatin and, obviously,

could perform extra-chromatin functions.

It has been reported in some publications that

free histones are present in the cell cytoplasm. Inpar-

ticular, already at the end of XX century Zlatanova

et  al.  [137] reported existence of a cytoplasmic pool

of H1 histones, mentioning, at the same time, that no

core histones were found in the cytoplasm. However,

recently information emerged on the presence

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of histones H3 and H4 in the cell cytoplasm, where

they bind with specific importins before being trans-

ported to the cell nucleus [138, 139]. Presence of main

histones (H1, H2A, H2B, H3, H4) in the composition of

plasma membranes has been mentioned in a number

of studies, where possible existence of additional ex-

tra-chromatin functions of histones has been suggest-

ed [127, 140, 141]. Moreover, histones were found in

mammals outside of the cells in blood flow, where they

play role of inflammation factors directly damaging

endothelial cells and also cells in various organs[142].

In general, it seems very likely that some histones

play not only structural and regulatory functions in

chromatin composition, but also could play various

roles outside of chromatin.

Non-histone chromatin proteins. The term non-

histone chromatin proteins (NHCP) has been suggested

more than 50 years ago to describe a large group of

proteins capable of binding chromatin, but differing in

structure and functions. Three families of high-mobil-

ity proteins are commonly assigned to NHCPs, which

are designated as HMGA, HMGB, HMGN (from high

mobility groups  A, B, N (see examples in [143-145]).

Proteins of these families are considered as structur-

al components and chromatin architectural factors;

however, they interact differently with genome DNA

and histones.

A number of proteoforms belong to the HMGA

family, which are products of expression of two genes

(HMGA1 and HMGA2). According to the data on P17096

and P52926 in the UniProt database, functioning of

each gene leads to formation of dozens of proteoforms

due to point mutations, alternative splicing, and post-

synthetic modifications. The available data indicate

that the corresponding proteins are capable of binding

with A  +  T-rich sites of genomic DNA, participate in

remodeling of chromatin, interact with transcription

factors, therefore providing regulation of expression

of different genes, and other.

It is important to note that the protein products

of the HMGA1 gene have been found in the nucleus

and nucleoplasm, as well as in cytoplasm (see P17096

UniProt).

Available data indicate that the proteins of HMGA

family perform their functions mainly in the course

of embryonic development, when expression of the

specified genes is sufficiently high, which is followed

by low expression in normal adult tissues or complete

absence of it [146]. However, proteins of the HMGA

family are present in significant amounts in the hu-

man tumor tissues, which is considered as a sign of

their involvement in the processes of carcinogenesis

and is associated with poor prognosis.

Accumulated information on the proteins of

HMGA family provide basis for designating them as

multifunctional proteins [147]. Moreover, it has been

discovered recently that the breast cancer cells secrete

the product of expression of the HMGA1 gene capable

of functioning as a growth factor outside of the cell;

this allowed to assign this protein to moonlighting

proteins [42].

Proteins of the family of human HMGB are encod-

ed by four genes (HMGB1, HMGB2, HMGB3, HMGB4),

expression of each of them results in formation of

several proteoforms. In particular, protein products

of the HMGB1 gene include four verified variants

with 26 post-synthetic modifications (P09429 UniProt).

They are localized in different cellular compartments

(nucleus, cytoplasm, cellular membranes, and others),

where they perform different functions. Moreover,

secretion of these proteins into extracellular medi-

um and their functioning there as a cytokine was

demonstrated. Respectively, according to a number

of authors, protein products of the HMGB1 gene are

moonlighting proteins [148].

Five genes encoding the HMGN proteins were

found in the human genome with their properties

described in the UniProt database (HMGN1 – P05114,

HMGN2– P05204, HMGN3– Q15651, HMGN4– O00479,

HMGN5 – P82970). Expression of the cited genes re-

sults in formation of several proteoforms due to

post-synthetic modifications. However, so far only two

isoforms determined by the HMGN3 gene were found.

Ithas been also observed that proteoforms determined

by the three genes are localized in different cellular

compartments: HMGN1, HMGN2 in cellular nuclei, nu-

cleoplasm and cytoplasm, and HMGN3– incell nuclei,

nucleoplasm, and mitochondria.

The HMGN proteins are synthesized in all cells

of vertebrates. During functioning they bind with

nucleosomes, as well as with the regulatory sites of

chromatin, including enhancers and promoters [144].

There are also direct data on multifunctionality of the

products of expression of the HMGN1 gene in mam-

mals [149].

Chromatin and transcription factors. At present

a large array of data has been accumulated on inter-

action of chromatin with hundreds of transcriptional

factors resulting in certain structural rearrangements

and changes in chromatin. Proteins capable of rec-

ognizing and binding to the specific DNA sequences

thus regulating transcription are considered as tran-

scription factors (see, for example, [123]). It has been

assumed that specificity of the transcription factor

binding to DNA is determined, on the one hand, by

the presence in their polypeptide sequence of specif-

ic DNA-binding domains, and on the other hand, by

availability in the polynucleotide sequences of DNA of

sets of related short sequences (motifs or sites) recog-

nized by the specific factors [123].

There are direct data demonstrating that cer-

tain transcription factors in addition to their canonic

MOONLIGHTING PROTEINS OF HUMAN AND SOME OTHER EUKARYOTES S49

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

functions have other functions. In particular, the tran-

scription factor ATF5 (AMP-dependent transcription

factor, ATF-5, according to Q9Y2D1 UniProt) capable

of stimulation or suppression of expression of certain

genes, is also required for realization of different stag-

es of mitosis. It participates as a moonlighting protein

in assembly of centrosomes, special organelles in eu-

karyotic cells [150].

Among the transcription factors there are pro-

teins with unordered structure capable of oligomer-

ization and interaction with other proteins or low

molecular weight ligands while performing their

functions (activation or repression of genes), such

as, for example, YY1 [151]. According to the available

data YY1 binds to the specific DNA motif present in

the regulatory elements of many genes. Respectively,

this factor is a pleiotropic regulator of a number of

cellular processes – cell proliferation, differentiation,

and apoptosis.

Furthermore, it has been shown that some of the

similar transcription factors, as well as individual sub-

units of oligomeric transcriptional complexes are in-

volved in the processes of carcinogenesis and, hence,

are characterized as oncoproteins [151, 152]. For ex-

ample, the human c-Fos protein, while localized in the

cell nucleus, forms a strong but non-covalent complex

with the transcription factor JUN/AP-1 and participates

in regulation of gene transcription (according to the

data on P01100 in UniProt). However, its presence was

observed also in endoplasmic reticulum and in cyto-

plasm. The c-Fos protein with non-nuclear localization

was shown to be able activating synthesis of cytoplas-

mic lipids in the cell of central nervous system and

support neuronal activity; hence, this provided a basis

for assigning it to moonlighting proteins [152].

The transcription factor EB (according to P19484

UniProt – Class  E basic helix-loop-helix protein  35,

TFEB, bHLHe35), which specifically recognizes and

binds the 5′-GTCACGTGAC-3′ sequences in DNA mole-

cules, is also assigned to moonlighting proteins. This

sequence is present in the regulatory sites of many

genes facilitating biogenesis and functioning of lyso-

somes. Obviously, nuclear localization of the transcrip-

tion factor EB is required for realization of its main

functions; however, significant fraction of this protein

is present in cytoplasm, where it is in composition of

the specific complex associated with lysosomes [153].

And finally, different data were summarized in a re-

cent review demonstrating that the transcription fac-

tor EB is capable of changing its localization, and, in

the process, it acts not only as a transcription factor,

but performs additional functions [38].

It could be stated in conclusion that many hu-

man proteins, which are in composition of chroma-

tin or interact with chromatin during functioning,

have additional functions. Some of them are already

considered as moonlighting proteins, and a number of

others could, likely, be considered as such based on

formal characteristics.

MOONLIGHTING PROTEINS

IN CELLULAR MEMBRANES

Modern notions on origins of life concentrate

specifically on the steps mediating transition from

prebiotic stage to formation of cellular forms due to

appearance of cell membranes (see, for example[154,

155]). It is assumed that the fundamental conditions

for this should be emergence of the source of amphi-

philic compounds capable of assembling into mem-

brane compartments. Consequently, model studies

are being conducted on self-assembly of simple mem-

branes of protocells as a prerequisite for evolution of

cellular metabolism and, practically, emergence of life

on the early Earth [155].

It is assumed that the membrane proteins em-

bedded into lipid bilayer (transmembrane proteins,

TMPs) play a vital role in membrane stability and for

performing many cellular functions, because they are

capable of interacting both with environment and in-

tracellular components. Polypeptide chains of TMPs

could be arranged in such a manner that they cross

the lipid bilayer only once (single-pass membrane pro-

teins), and, as a consequence, their N-terminal part is

exposed the extracellular medium, while the C-termi-

nal– to cytoplasm (typeI) or vice versa (typeII) [156].

TMP with their polypeptide chain spanning the lipid

bilayer two or more times are also known (multipass

membrane proteins) [156, 157].

Polypeptide chains of the TMPs designated as

single-pass membrane proteins usually contain sev-

eral domains with each of them performing its own

specific function. In other words, these proteins are

multifunctional, but based on the existing nomencla-

ture they are not assigned to moonlighting proteins.

At the same time, it has been demonstrated that some

multipass membrane protein are localized not only in

the structure of cellular membranes, but also in cy-

toplasm, such as, for example, epithelial membrane

protein  2 (EMP2) according to the data on P54851 in

UniProt. Moreover, it has been noted that this small

protein in addition to membrane functions have other

functions, in particular, associated with regulation of

functioning of some signalling pathways and involve-

ment in carcinogenesis [158].

Among the transmembrane proteins character-

ized as moonlighting proteins, some attention has

been drawn to the so-called ectoenzymes, which are

membrane-bound enzymes with a catalytic center lo-

cated outside the cell [23]. In particular, it is known

that one of such enzymes, aminopeptidase N (CD13),

SHISHKINS50

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

which is a Zn

-dependent metalloproteinase catalyz-

ing hydrolysis of various peptides, also could play a

role in amino acid transport, participate in angiogen-

esis, and serve as a receptor of human coronavirus

(see information on P15144 in UniProt). Human TMPs

that are Zn

transporters, which are capable of per-

forming additional functions, have been also consid-

ered as moonlighting proteins [159, 160]. It has been

noted that the Zn

transporters in mammals including

humans, comprise a rather large family of proteins

formed in the process of long evolution with repre-

sentatives of this protein family located in different

cellular membranes. They are capable of forming

oligomeric complexes participating in numerous sig-

naling processes. More than ten genes encoding Zn

transporters were identified in the human genome.

Examples of diversity of properties and cellular lo-

calization could be found in the UniProt database for

the Zn

transporter ZnT-1 (synonym– Proton-coupled

zinc antiporter SLC30A1, Q9Y6M5 in UniProt).

In could be stated in conclusion that considering

the fact that membrane proteins could comprise more

than 30% of all human proteins (according, for exam-

ple, to[156]), it is likely that the available information

on those that should be considered as moonlighting

proteins will increase.

SPECIALIZED DATABASES

ABOUT MOONLIGHTING PROTEINS

Accumulation of knowledge on moonlighting pro-

teins in the second decade of XXI century, which was

accompanied with the development of postgenomic

science and bioinformatics, motivated creation of spe-

cialized databases for moonlighting proteins.

In particular, in the end of 2014 a work has been

submitted for publication by the scientists from Uni-

versity of Illinois (USA) describing the specialized da-

tabase containing information of moonlighting pro-

teins named MoonProt. This work was published at

the beginning of 2015 [161]; it included information

on 200 experimentally verified moonlighting proteins.

It is important to mention that among the authors of

this paper was Constance  J.  Jeffery. Later versions of

the MoonProt (version 3.0) contained annotations on

the properties of more than 500 moonlighting protein

from different organisms [35].

Practically simultaneously with the American sci-

entists, a group of researchers from Spain created and

used another database named MultitaskProtDB, which

contained summarized data on several hundreds of

know moonlighting proteins [36,  162]. According to

the opinion of the developers, the MultitaskProtDB

opened the possibilities for identification of such pro-

teins through analysis of their structural and func-

tional characteristics from numerous other databas-

es with the help of bioinformatics. The version of

MultitaskProtDB-II from 2018 contained information

on 694 moonlighting proteins [162]. The team of re-

searchers that created this database included scien-

tists from Uruguay in addition to the researchers from

Spain. The list of sources analyzed by the creators of

the MultitaskProtDB-II database has been expanded

significantly. Moreover, it included new information

from the publications presented in the PubMed da-

tabase, as well as in the new versions of the UniProt

database. The developers paid special attention also

to the information from a number of other databases

including the Online Mendelian Inheritance in Man

(OMIM), Human Gene Mutation Database (HGMD), the

Therapeutic Target Database (TTD), ands the DrugBank

database. The list of the used bioinformatics tools also

was expanded.

In the second decade of XXI century a specialized

database was created in France on Extreme Multifunc-

tional proteins in human including moonlighting pro-

teins, which was named MoonDB [163, 164]. In this

work special attention was paid to the studies devot-

ed to investigation of protein–protein interactions,

which produced new data on specific interactomes of

moonlighting proteins. As a consequence, the develop-

ers identified 430 extremely multifunctional proteins

combining various network information with informa-

tion from annotations of the proteins form different

databases (including, for example, OMIM NCBI). The

second version of the MoonDB 2.0 database (http://

moondb.hb.univ-amu.fr/) included information not

only on the human moonlighting proteins, but also on

similar proteins from some eukaryotes with genomes

completely sequenced [164]. In addition, interface of

the second version of MoonDB was completely rede-

signed and improved, and the existing annotations

were cross-referenced with the UniProt database.

Next, publication of the Chinese researchers should

be noted, which developed a specialized PlantMP da-

tabase that include information on moonlighting pro-

teins in plants [165]. This database included materials

on 110 known moonlighting proteins in plants, as well

as on 10 proteins considered to be likely moonlighting,

and 27 of presumed moonlighting proteins. PlantMP

uses identifiers and designations from the UniProt da-

tabase, which opens up the possibilities for searching

canonical and additional functions of the proteins.

Moreover, the PlantMP database also presents refer-

ences to the publications in the PubMed database.

The developers expressed their opinion that organi-

zation of all materials on the moonlighting proteins

of plants on one platform would help the research-

ers to collect both processed and unprocessed data on

particular plants including information on molecular

functions and structural features that are required

MOONLIGHTING PROTEINS OF HUMAN AND SOME OTHER EUKARYOTES S51

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

to formulate a hypothesis in fundamental and applied

science and for biotechnological innovations.

And finally, recently another report has ap-

peared on the specialized database developed in Italy

named MultifacetedProtDB, which included extensive

information on human moonlighting proteins [166].

This new database included information not only

on moonlighting proteins, but also on many other

multifunctional proteins. Obviously, the Multifacet-

edProtDB could allow optimizing investigations of

moonlighting proteins and multifunctional proteins.

The MultiFacetedProtDB can be accessed at: https://

multifacetedprotdb.biocomp.unibo.it/.

It seems that creation of various databases inte-

grating existing information on moonlighting proteins

could open wide opportunities for targeted investiga-

tion of these proteins in different multifactorial bi-

ological processes such as signaling and metabolism

regulation, gene expression, and cell–cell communi-

cations, which is extremely important for elucidation

of pathogenesis of many diseases.

CONCLUSIONS

The presented analysis indicates that, at present,

investigation of moonlighting proteins comprises a

separate scientific area examining issues of both the-

oretical and applied nature associated, for example,

with diagnostics of socially significant diseases.

Thus, for theoretical biochemistry, of consider-

able interest is the formation of ideas about due to

what, when and how special multifunctional proteins

appeared, consisting of one polypeptide chain, but

possessing several functions, which in their structure

do not have different domains to provide these func-

tions, i.e., moonlighting proteins. One of the possible

explanations could be information on intrinsically un-

ordered proteins, whose tertiary structure capable of

significant changes depending on environmental con-

ditions. The phenomenon of switching functions in

moonlighting proteins at least in some cases is likely

associated with this type of changes. However, it re-

quires further verification whether this mechanism is

universal or widespread.

It seems important to discuss theoretical issue

on evolution of additional functions in moonlight-

ing proteins. Already in the early publications (for

example, [10]) the opinion has been expressed that

additional functions could be the result of molecu-

lar evolution. However, an alternative point of view

also exists, according to which ancient proteins were

intrinsically multifunctional, and evolution resulted

in emergence of proteins with the single specialized

functions. This point of view is shared by the author

of this paper.

Many studies on moonlighting proteins that are

considered as a special group of multifunctional pro-

teins focus on investigation of molecular basis of

pathogenesis of widespread diseases including malig-

nant tumors. It is assumed that potential prognostic

markers and/or molecular targets could be identified

as a result, which could be used for the development

of novel effective treatment strategies. For similar

purposes, in several countries (USA, Spain, France,

China, Italy) have created and are using specialized

databases containing information about moonlighting

proteins.

In conclusion, it should be stated that the collect-

ed information could provide promise that investiga-

tion of moonlighting proteins would expand and in

our country in the nearest future, which could facil-

itate creation of the specialized database in Russia.

Funding. This work was supported by ongoing in-

stitutional funding. No additional grants to carry out

or direct this particular research were obtained.

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The author of this work de-

clares that he has no conflicts of interest.

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