Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, Nos. 12-13, pp. 2287-2291 © Pleiades Publishing, Ltd., 2024.

2287

HYPOTHESIS

Induction of IMPDH-Based Cytoophidia by a Probable

IMP-Dependent ARL13B–IMPDH Interaction

Najva Alijani

and Razieh Yazdanparast

1,a

Institute of Biochemistry and Biophysics, University of Tehran, 13145-1384 Tehran, Iran

e-mail: ryazdan@ut.ac.ir

Received August 26, 2024

Revised October 22, 2024

Accepted November 18, 2024

Abstract— Inosine Monophosphate Dehydrogenase (IMPDH) catalyzes rate-limiting step of the reaction con-

verting inosine monophosphate (IMP) to guanine nucleotides. IMPDH is up-regulated in the healthy prolif-

erating cells and also in tumor cells to meet their elevated demand for guanine nucleotides. An exclusive

regulatory mechanism for this enzyme is filamentation, through which IMPDH can resist allosteric inhibition

by the end product, GTP. It has been proven that intracellular IMP, which rises during the proliferative state,

potentially promotes IMPDH filamentation. On the other hand, interaction of IMPDH with ADP-ribosylation

factor-like protein 13B (ARL13B) directs guanine biosynthesis toward the denovo pathway. However, ARL13B

is not localized in the IMPDH-based cytoophidia, super structures composed of bundled IMPDH filaments and

other proteins. Here, we hypothesized that ARL13B could increase availability of the de novo-produced IMP

for IMPDH by interacting with the IMP-free IMPDH and microtubules adjacent to the purinosome. Following

IMP-binding, IMPDH would be released from ARL13B and preferentially associated with its cytoophidia. Con-

sidering clinical side effects of catalytic inhibitors of IMPDH and their ability to induce IMPDH cytoophidia,

we suggest that combination of proper doses of IMPDH catalytic inhibitors and inhibitors of the denovo IMP

biosynthesis could be more effective in controlling cell proliferation.

DOI: 10.1134/S0006297924120162

Keywords: ARL13B–IMPDH interaction, IMP, cytoophidia, purine biosynthesis

Abbreviations: ARL13B, ADP-ribosylation factor-like protein

13B; GTP, Guanosine-5′-triphosphate; IMP,inosine-5′-mono-

phosphate; IMPDH, inosine monophosphate dehydrogenase .

* To whom correspondence should be addressed.

INTRODUCTION

Inosine monophosphate dehydrogenase (IMPDH),

a critical enzyme in purine metabolism, catalyzes

rate-limiting step in the de novo biosynthesis of gua-

nine nucleotide by conversion of inosine-5′-monophos-

phate (IMP) to xanthosine-5′-monophosphate (XMP)

[1]. While its inhibitors are clinically used as immu-

nosuppressive, antiviral, and antimicrobial drugs, they

have not yet been approved for cancer treatment. This

is mainly due to severe side effects at high clinical

doses and inconsistent responses [2], which highlights

the need for further IMPDH research.

In vertebrates, octameric units of IMPDH can

reversibly assemble into single-stranded filaments,

which, in turn, can associate to create intricate struc-

tures known as “cytoophidia”, also known as “rods

and rings” (RR) structures because of their shapes

[3-5]. This fascinating ability adds an extra level to

the enzyme activity regulation, facilitating resistance

to allosteric inhibition by guanosine-5′-triphosphate

(GTP)  [6]. Some conditions, such as a high sub-

strate-to-product (IMP/GTP) ratio [6, 7], cell treatments

with IMPDH inhibitors such as Mycophenolic acid

(MPA) and/or Ribavirin [3, 4], and deficit of essential

purine biosynthetic precursors such as glutamine and

folate [8, 9], promote cytoophidia formation. On the

other hand, some highly proliferative cells, such as

induced pluripotent stem cells (iPSCs), mouse embry-

onic stem cells (ESCs), and mitogen-activated T-cells

robustly form cytoophidia in rich media without ex-

ternal treatments [4, 7, 10], probably due to the elevat-

ed IMP level resulting from upregulated proliferation

signals [6].

ALIJANI, YAZDANPARAST2288

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

HYPOTHESIS AND SUPPORTING DATA

In tumor cells, the rate of GTP metabolism under-

goes more extensive changes than that of ATP. While

ATP levels show a modest rise of approximately 20%

in tumor cells, GTP levels significantly increase by

nearly 200% relative to normal cells [11, 12]. Interest-

ingly, upregulated GTP is redirected toward nucleolus

for tRNA and rRNA synthesis, the most abundant RNA

species in the cell, which have an indispensable role

in protein synthesis, resulting in nucleolar transforma-

tion and cell proliferation [13]. Dramatic upregulation

of GTP in the tumor cells could suggest preferential

direction of IMP toward GTP biosynthesis rather than

ATP production. We speculate that the GTP channel-

ing toward nucleolus as well as a probable direction

of IMP toward IMPDH would increase the IMP/GTP

ratio in the surrounding environment of the IMPDH

and therefore, could lead to IMPDH filamentation.

Moreover, constitutive formation of IMPDH-based cy-

toophidia in some highly proliferative cells might be

a consequence of not only a balance between IMP

production and GTP consumption but an equilibrium

between the probable direction of IMP toward IMPDH

and GTP channeling toward nucleolus.

While IMP biosynthesis in quiescent cells is low

and is realized through the salvage pathway, the high-

ly proliferative cells mainly meet their high demand

for purine nucleotides via the de  novo synthesis path-

way [14,  15]. Notably, interaction of IMPDH with the

small GTPase from the ARF family, named ADP-ribo-

sylation factor-like protein 13B (ARL13B), shifts pu-

rine biosynthesis from the salvage pathways toward

the de  novo pathways [16]. Despite the enrichment

of ARL13B in primary cilia as the cilium marker, lit-

erature also documents presence of Arl13B in cyto-

plasm associated with biological functions [17,  18].

Cytoplasmic ARL13B colocalizes and cooperates with

some cytoskeleton components involved in endocytic

recycling traffic such as Arf6, Rab22, CD1a, actin, and

also microtubules [17]. Interaction of the cytoplasmic

ARL13B with cytoskeleton components also regulates

cell migration [18]. Distribution of ARL13B between

the cilia and cytoplasm as well as its stability and

functions could be affected by various factors. For in-

stance, lipid modifications, especially palmitoylation,

are vital for ciliary localization, function, and stability

of ARL13B [19]. Based on the present literature data

on disappearance of the cilia body structure from the

cell surface during the processes of cell division and

migration [20-22], it is assumed that the ARL13B con-

tent could probably end up in the cytoplasm. Inter-

estingly enough, our preliminary data confirmed this

prediction.

Shireman et al. showed that the 96-h treatment

of cells with Temozolomide (TMZ), an alkylating agent,

increases ARL13B expression and its interaction with

IMPDH outside of the primary cilia in glioblastoma

tumor cells  [16]. It was also determined that the ef-

fects of TMZ on purine metabolism are mediated by

the ARL13B–IMPDH interaction and are independent

of the Sonic hedgehog signaling(Shh) and primary cil-

ia. The authors hypothesized that by shifting purine

metabolism from the salvage pathway to the de  novo

pathway, this interaction could prevent the cell from

using alkylated GTP and maintain cell survival  [16].

However, it is unclear how the ARL13B–IMPDH inter-

action could cause this switching in the purine bio-

synthetic pathway. It can be seen from the particu-

lar results of this study that the 96-h treatment with

TMZ can increase incorporation of the radio-labeled

hypoxanthine and glycine into DNA and RNA. This

means that the ARL13B–IMPDH interaction could in-

crease incorporation of the precursors belonging to

two IMP-dependent pathways of purine metabolism:

the hypoxanthine-dependent salvage pathway and the

de  novo pathway (Fig.  5E from [16]). However, TMZ

treatment, and subsequent rise in the ARL13B–IMPDH

interaction, decrease the GMP production from the hy-

poxanthine-dependent salvage pathway in the tumor

sample (Fig.  5F from[16]). These data suggest that the

TMZ-induced increase in incorporation of

H-hypoxan-

thine into nucleic acids (Fig.  5E from [16]) is proba-

bly due to the increased conversion of hypoxanthine

to AMP only and not to GMP. This suggestion is con-

firmed by the increase in the downstream product of

AMP catabolism, hypoxanthine, in the TMZ-treated

samples (Fig.  5F from [16]). Decrease in the fraction-

al enrichment of hypoxanthine-derived IMP and GMP

in the TMZ-treated samples (Fig. 5G from [16]) also

suggests direction of the hypoxanthine-derived IMP to-

ward the AMP synthesis and not GTP. Our conclusions

from Fig. 5 of [16] are summarized in Scheme  1.

Accordingly, increase in the ARL13B–IMPDH in-

teraction directs the de novo-produced IMP toward

IMPDH, theoretically supporting cytoophidia forma-

tion. In this scenario, ARL13B–IMPDH interaction and

cytoophidia formation, as two probable aspects of GTP

metabolic reprogramming in proliferating cells, could

occur in the same spatiotemporal pattern. Our lab-

oratory has detected increase of the ARL13B–IMPDH

interaction in the cytoophidia-forming cells and its

positive correlation with the cell viability. We believe

that the ARL13B–IMPDH interaction could change ori-

entation and proximity of IMPDH toward purinosome,

a metabolon consisting of the enzymes responsible for

the de  novo purine biosynthetic pathway. This assump-

tion emerged from the summation of three indepen-

dent observations. First, purinosome moves along the

microtubule toward mitochondria and localizes in the

mitochondria-microtubule interface at the peak of pu-

rine demand [23]. Second, there is evidence of ARL13B

INDUCTION OF IMPDH-BASED CYTOOPHIDIA 2289

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Scheme 1. Separation of IMPDH from the IMP-dependent salvage pathway of purine biosynthesis and increasing its ac-

cess to the de  novo-produced IMP after 96-h TMZ treatment of glioblastoma cells (GBMs). Purinosome, a metabolon of the

enzymes participating in the de  novo purine biosynthetic pathway (blue pathway), and hypoxanthine-guanine phosphori-

bosyltransferase (HGPRT), an enzyme responsible for recycling purine from hypoxanthine (yellow pathway) and guanine

(not shown here), provide the cellular IMP pool. IMPDH and adenylosuccinate synthase (ADSS) jointly use this IMP re-

source to produce GMP and AMP, respectively. Increased ARL13B–IMPDH interaction following 96-h TMZ treatment

drives IMPDH to the de  novo-produced IMP and prevents its access to the salvage-produced IMP. This means a decreased

role of the IMPDH’s competitor, ADSS, in using the de  novo-produced IMP and its greater role in using the salvage-pro-

duced IMP. ADSS, adenylosuccinate synthase; Glu and AA, glucose and amino acids as precursors of de  novo biosynthesis

of IMP; HX, hypoxanthine; HGPRT, hypoxanthine-guanine phosphoribosyltransferase.

and microtubule association in the cytoplasm, though

lower than in cilia. ARL13B is a membrane-associated

and anchor-like protein whose interaction with axo-

neme tubulin is vital for the uniform distribution of

the ciliary membrane proteins  [24]. ARL13B margin-

ally co-localizes with microtubules in the cytoplasm

and can participate in endosomal recycling along mi-

crotubules through interaction with actin  [17]. Fur-

thermore, some studies demonstrated participation

of ARL13Bs in other microtubule-associated processes

such as cell migration and proliferation [18,  20], which

typically happen in the temporary absence of cilia

[20-22]. Third, cytoophidia is associated with some in-

tracellular membranes such as mitochondrial and en-

doplasmic reticulum (ER) membranes [25]. Moreover,

another consistent component of the IMPDH-based

cytoophidia, ADP-ribosylation factor-like protein  2

(ARL2), is involved in both αβ-tubulin biogenesis and

mitochondrial fusion [25]. These findings could imply

a relationship between the location of cytoophidia and

purinosome in the cells or even their co-localization

in the proximity of intracellular membranes with the

emergence of high demand for GTP. This relation be-

comes feasible with the help of tubulin-interacting

small GTPases like ARL2 and ARL13B. In our model,

the ARL13B–IMPDH interaction anchors IMPDH to the

microtubule and in proximity to purinosome. Since

it was reported in the previous study that ARL13B is

not localized in the IMPDH-based cytoophidia [25],

we believe that ARL13B only interacts with soluble

and IMP-free-IMPDHs. IMP binding triggers dissocia-

tion of IMPDH from ARL13B, making it free to form

cytoophidia. This model could explain the role of

ARL13B–IMPDH interaction in the preferential direc-

tion of the de  novo-synthesized IMP toward IMPDH for

robust GTP production.

Induction of the ARL13B expression and ARL13B–

IMPDH interaction by TMZ [16] as an alkylating agent

suggests that the recognized GTP deficit might increase

the ARL13B–IMPDH interaction. As two GTP-binding

proteins, ARL13B and/or IMPDH could directly sense

the level of GTP. Other GTP-sensing proteins such as

phosphatidylinositol 5-phosphate 4-kinaseβ (PI5P4Kβ),

a cytoplasmic membrane-bound protein with a unique

GTP-dependent kinase activity [26], could also partic-

ipate in any step of IMP channeling toward IMPDH

upon GTP depletion. However, according to the above

assumption, IMPDH inhibitors could induce the

ARL13B-IMPDH interaction by reducing GTP levels.

The level of this interaction would depend on the

amount of IMPDH proteins with an empty active site

and, therefore, on the inhibitor concentration. Lower

concentrations of IMPDH inhibitors, with sufficient

enzymes having empty active sites, could induce

ARL13B–IMPDH interaction and cytoophidia forma-

tion. In this case, they can compensate for GTP re-

duction and even induce cell growth by producing a

high level of GTP. In contrast, lethal doses of inhib-

itors could decrease the ARL13B–IMPDH interaction

by depleting the enzyme population with an empty

ALIJANI, YAZDANPARAST2290

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

active site. Although lethal doses could be successful

for cancer control in  vitro, their clinical use is not fea-

sible due to severe side effects. Hence, it seems that

inhibitors of the de  novo IMP biosynthesis, which are

theoretically cytoophidia-dissembling agents, could be

useful in this case. They could not only decrease the

level of the IMPDH substrate, but could also decrease

cytoophidia formation. Thus, a combination therapy of

these two types of inhibitors could be more effective

in controlling tumor cells than the IMPDH inhibitors

alone. This hypothesis is under investigation in our

laboratory and the results will be published soon.

Acknowledgments. The authors appreciate the

joint financial support of this investigation by the

Research Council of the University of Tehran and the

Iranian National Science Foundation (INSF).

Contributions. Both authors contributed to the

study’s conception and design. N.A. performed mate-

rial preparation, data collection, and analyses. N.A.

also prepared the first draft of the manuscript, which

was edited by R.Y. Both authors read and approved the

final draft to be submitted.

Funding. The Research Council of the University

of Tehran and the Iranian National Science Founda-

tion (INSF) have provided support for this investiga-

tion.

Ethics declarations. This work does not con-

tain any studies involving human and animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

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