Molecular interactions between G-actin, DNase I and the beta-thymosins in apoptosis: a hypothesis

Medical Hypotheses I Medicd Hypothaes (1924) 43. 125-131 0 Longman Group LuJ 19Y4

Molecular Interactions Between G-Actin, DNase I and the Beta-Thymosins in Apoptosis: a Hypothesis A. K. HALL Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 lC?J, lJH

Abstract - The beta-thymosins are a family of <5kDa (MW), mostly acidic, proteins which were originally defined in the immune system. Recently, specific members of this family of cytoplasmic polypeptides, namely beta-4 and beta-lo, were shown to bind monomeric G-actin both in vitro and in vivo. Whilst many aspects of programmed cell death or ‘apoptosis’ remain to be defined, the Ca2+/Mg2+-dependent endonuclease, DNase I does feature in this process. Monomeric G-actin binds to and inhibits the DNA-degrading activity of DNase I. Given that the intracellular abundance of thymosins beta-4 and beta-10 is related to cell division and differentiation and that anticancer/morphogenic agents such as retinoic acid (RA) and cyclic AMP modulate expression of their respective genes, it is possible that these G-actin sequestering proteins play significant roles in apoptosis perhaps mediated via DNase I.

Introduction Elimination of specific cell-types during the course of embryogenesislmorphogenesis can be achieved by an active process of programmed cell death or suicide commonly termed ‘apoptosis’ - a name derived from the Greek word for leaf abscission. A number of highly characteristic morphological signs herald the onset of apoptosis, the most salient of which are, a diminuation in cell volume, dilation of the endoplasmic reticulum and the condensation of chromatin into conspicuous nuclear membraneassociated dense bodies (l-3). Biochemically, apoptosis can also be recognized by the evolution of nucleosomal DNA fragments which form a very distinctive ladder as detected in ethidium bromidestained agarose gels (4-6). Although others exist, one endonuclease principally responsible for DNA degradation during apoptosis is currently thought to be

deoxyribonuclease I (DNase I) (7). Whilst overexpression of DNase I in transfected COS cells certainly mimics the DNA degradation scenario (symptom?) observed during natural apoptosis (7), the recent revelation that the variable regions [V3 and V13] of the 28s ribosomal RNA can also undergo selective cleavage during early apoptosis (8) suggests that the Ca2+Ng2+-dependent (9-11) DNase I may not be the only endonuclease participant in this suicide mechanism. This caveat is further corroborated by the observation that zinc [Zn2+] inhibition of DNase I fails to curtail the formation of large DNA fragment formation as induced by dexamethasone in apoptotic thymocytes whilst formation of low MW oligonucleosomal fragments continues (12). Thus, DNA degradation during apoptosis, whilst seemingly not an absolute requirement (13), is a multistage process, the initial phases of which, involve synthesis of high MW [>700, 300, 50 Kbp] DNA fragments followed by a

Date received 2 April 1993 Date accepted 17 May 1993

125

126 DNase I-mediated [Zn2+-sensitive] catabolic genesis of low MW 180-200 bp oligonucleosomal fragments. It is possible therefore, that initial phases [
MEDICALHYPOTHESES

its homology to beta-4, might be expected to fulfill similar roles within the cell. The beta-10 gene has been shown to be developmentally-regulated during neuroembryogenesis (69-72) as well as in the human kidney (73). Retinoids can modulate expression of beta-10 (but not beta-4) in a number of cell lines (74-76). The thymosin beta-10 gene is sensitive to inhibition by cyclic AMP and serum deprivation (77) - two things known to induce apoptosis. Molecular cloning of the thymosin beta-10 gene and functional analysis of its promoter reveal the presence of several putative response elements sensitive to retinoid receptors (RAR, RXR) (78,79), whilst other studies indicate that at least one nuclear retinoic acid receptor can target the endogenous gene (80). Interestingly, expression of both beta-4 and beta- 10 genes is avidly associated with active cell proliferation, in fact, expression of these two beta-thymosin genes is conspicuously enhanced in many tumors (73). Other workers have gone on to show that levels of the beta-4 protein are regulated during specific phases of the cell-cycle in cultured rat thymocytes (81,82) - specifically, Schobitz et al demonstrated that this thymosin increases IO-fold during the Gl phase. Recently, Safer et al (83,84) showed that thymosin beta-4 and Fx, an actin-sequestering peptide are indistinguishable. Indeed, beta-4 contains a 9-residue sequence [LKKTETQEK, residues 17-251 that is believed to be the active G-actin binding domain (85) similar motifs appear in other actin-binding proteins such as actobindin. More recently, microinjection of synthetic thymosin beta-4 into 3T3 cells was found to induce a depolymerization of actin filaments (F-actin) (86). The sequence [LKKTETQEK] also appears in the beta-10 peptide sequence (68) and so it is no surprise that the thymosin beta-10 molecule can also sequester/bind G-actin monomers (87,88). By virtue of its relatively high intracellular concentration (500600 pM in platelets), it subsequently transpired that beta-4 is a major intracellular actin-binding agent indeed, most of the sequestered monomeric G-actin pool appears to be present as the beta-6complexed form (90). So the stage is therefore set for a molecular interaction between actin-binding proteins such as thymosin beta-4/bets- 10, monomeric G-actin and DNase I. Actin and DNase I There can be little doubt that the 42kDa (MW), 375 residue actin molecule plays a pivotal role in the biology of the cell. Actin is the most abundant protein in mammalian cells (91) and this entity is an integral part of multiple pathways operative during normal and abnormal cell function. G-actin polymerizes to

G-ACTIN. DNASE I AND THE BETA-THYMOSINS

IN APOPTOSIS

form filaments in the presence of mono [K+] and divalent [Ca2+, Mg2+] cations and Pollard and Cooper (92) have provided a comprehensive review of this 4-step, ATP-dependent G-actin polymerization mechanism. Actin polymerization/depolymerization exists in a state of dynamic equilibrium which is dependent upon many variables including the ambient levels and proximity of quite a few actin scavenging proteins. Listed in this category are proteins such as vinculin, filamin, fodrin, myosin, tropomyosin, alphaactinin, fimbrin (F-actin crosslinker), villin/gelsolin (actin cleavers), profilin and DNase I. Of the Gactin binding proteins, DNase I, in the form of the actin-complex is to date the only complex to have been studied at the level of atomic resolution (93,94). Monomeric G-actin binds to and almost completely inhibits the nucleolytic activity of DNase I by formation of a high affinity stoichemetric (1: I ratio) complex (95) - this property (actin-inhibition of DNase I) is a highly diagnostic feature of this apoptotic endonuclease (96). It is of interest that in addition to its overt nucleolytic capacity, DNase I can itself inhibit (97) and actively induce (98) actin depolymerization. That actin and profilin (and beta-4/bets-10 and possibly other beta-thymosin ?) can bind simultaneously to DNase I suggests the presence of multiple (spatially distinct) binding sites for these cytoskeletal elements on the DNase I molecule. It could be argued that the actin-DNase I binding/inhibition phenomenon might just be coincidental or it might represent a mechanism that plays a significant role in apoptosis. For its part, the 42kDa (MW) actin molecule can play functional roles quite distinct from its primary function as an intracellular structural/cytoskeletal element. Actin is, for example, toxic when exuded from dead or dying cells into the extracellular space (reviewed, 99). Beta-thymosins,

DNase I, G-actin and apoptosis

Taking these facts/observations into consideration it is possible to envisage a scenario (Fig.) whereby these molecules can interact to influence cell-cycle related events such as apoptosis. At present, no hard data is available that directly implicates the beta-thymosins as playing an active role in programmed cell death. Nevertheless, several experimental observations imply a possible link between beta-4/bets-10 and apoptosis. Firstly, the G-actin sequestering thymosin beta10 can be induced by retinoic acid (RA) in neuroblastoma cells (NB) (74,75) and RA treatment of NB cells is positively associated with increased signs of apoptosis (100). Secondly, thymosin beta-4 (101) and beta-10 (102) are regulated during the formation and functional demise of the ovarian corpus luteum

127 (CL). Thirdly, these oscillations in beta-thymosin gene expression coincide with both spontaneous and prostaglandin (PG)-induced apoptosis (103). 1.n fact, induction of beta- 10 gene expression (I 02) parallels the PG-stimulated increase in ovarian CL oligonucleosome formation (103). Fourthly, direct interference with intracellular (episomal) thymosin beta-10 gene expression either via use of antisense deoxyoligonucleotides (104) or by overexpression (or underexpression) enforced by eukaryotic expression vectors (105) seems to modulate the rate of cell proliferation. In the scheme (Fig.) I present the (perhaps overly simplistic) hypothesis that by virtue of their ability to sequester G-actin, the beta-thymosins (and perhaps other G-actin binding proteins?) might actively influence a cell’s destiny. Certainly, the DNase I inhibitory proclivity of G-actin can be compromised by beta4/beta- IO. Perhaps via sequestration, beta4/bets-10 neutralizes the inhibitory flirtation between monomeric G-actin and DNase I which is then ‘free’ to participate in programmed cell death. However, enthusiasm for this concept is somewhat tempered by the fact that within the cell, the lysosomal DNase I is, by definition, compartmentally segregated (92) from the normally extranuclear/cytoplasmic G-actin/betathymosin proteins (106,107) - though this geographic isolation is altered during the ‘structural chaos’ that characterizes apoptosis (and transformation). Apoptosis and cell proliferation are two phenomena that can coexist, that is, rapid cell division is accompanied by vigorous cell death. This is especially true in malignant tumors where beta-thymosins are overexpressed (73), where apoptosis appears to be the most significant mechanism for cell death (108) and wherein elements of the cytoskeleton, such as filamentous actin, are not only less well defined, but are also less abundant. From a developmental perspective, G-actin and the beta-thymosins may conspire to regulate programmed cell death. For example, both beta-4/bets-10 are highly expressed during the early phases of neuroembryogenesis - a period characterized by rampant cell proliferation (109). It is also a fact that the dynamics of actin polymerization change during neural maturation - where G-actin prevails in the embryonic CNS and F-actin is more pervasive in the adult tissue (110). Conspicuously absent from the scheme (Fig.) are the multitude of other G-actin binding proteins as well as additional unidentified and identified thymosin-binding proteins such as equine leukocyte elastase inhibitor (I 11). Teleologically, sequestration of G-actin monomers could represent a mechanism by which cells are trapped in the Gl/GO phase and/or persuaded to take the suicide option via an ‘unleashing’ of DNase I or

128

MEDICAL HYPOTHESES

ACTIN-THYMOSIN INTERACTIONS ONCOGENESIS & DIFFERENTIATION F-ACTIN FORMATION CONSEQUENCES

CONSEWENCES -

camel !NHrnrrlOEl NEURITCGENESIS’

SEQUESTRATION

. .

SIGNALS EXTRACELLULAR

Fig. Tentative mechanism tiation and apoptosis.

by which retinoid-responsive

thymosin beta-10 might influence the cell cycle phenomena

the genes/signals that initiate apoptosis. The G-actin binding beta-thymosins might play a pivotal regulatory role at this cell-cycle checkpoint. The ‘keys’ to the ‘cell-life lock’, namely the differentiationapoptosis inducing agents such as cyclic AMP and RA modulate the beta-10 gene (74-76) may thus represent one mechanism by which such signals counter unrestrained cell division. Questions,

questions,

questions

Taking a stop backwards and peering into the expansive, mostly uncharted murky depths of the cell, it is dauntingly obvious that a veritable cornucopia of forces, seen and unseen are involved in apoptosis and the beta-thymosins might only represent a trivial element(s) of this obfucated farrago. A flotila of important questions need to be addressed including:

(9 (ii) (iii) (iv)

Do retinoids (vitamin D/thyroxine) target the bcl-2 gene (during development)? Do thymosins beta-4/bets-10 (via G-actin sequestration) stimulate apoptosis? Does the bcl-2 protein influence G-actin polymerization? Does the bcl-2 protein bind beta-4? (A putative beta-4 binding motif ATAGT [III] does appear in the bcl-2 sequence [ATAGP]). Is there a bcl-2 gene family? Is unsequestered G-actin per se an antiapoptotic signal?

(vii) What is signal(s)?

(are)

the

initial

of mitosis, differen-

cell

suicide

The cell cycle conundrum has many loose ends, perhaps the cytoskeleton literally ties it all together? Whilst many mysterious aspects of the cell’s private life will remain beyond the scope of human understanding for some time, interest in the ‘apoptosis story’ is certain to accelerate rapidly (due to its implications for our understanding of neoplasia) and, as each page is turned, our collective perception of the ‘cell-cycle’ might ultimately have to be reconceptualized to embrace the notion of the 3-way struggle (choreographed by Ca2+, oncogenes, tumor suppressors) between mitosis, differentiation and cell death. Acknowledgements I thank Ann Hall for typing this manuscript. Alan Hall is funded by NC1 CA494222-04 and C R Bard Inc. Supported by NIHDK4758802. References Hincliff JR, IN: Bowen ID, Lockshin RA, eds. Cell death in biology and pathology. London: Chapman and Hall. 1981: 3578. Gerschenson LE, Rotello RJ. Apoptosis: a different type of cell death. FASEB J 1992; 6: 245tX2455. Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin

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