Date sent:      	Mon, 22 Oct 

Interactions Between Rnase L Ankyrin-Like Domain and ABC Transporters as
a Possible Origin for Pain, Ion Transport, CNS and Immune Disorders of
Chronic Fatigue Immune Dysfunction Syndrome

J of Chronic Fatigue Syndrome, Vol. 8. No. 3/4. 2001. pp. 83-102

Patrick Englebienne, PhD; C. Vincent Herst, PhD; Karen De Smet,
PhD; Anne D'Haese, MS; Kenny De Meirleir, MD, PhD

Patrick Englebienne is affiliated with the Université Libre
de Bruxelles (Brugmann Hospital) and is Consultant, RED Laboratories,
Brussels, Belgium.
C. Vincent Herst, Karen De Smet and Anne D'Haese are affiliated with the
R&D Department of RED Laboratories, Brussels, Belgium.
Kenny De Meirleir is affiliated with the Vrije Universiteit Brussel,
Brussels, Belgium.
Address correspondence to: Patrick Englebienne, PhD, RED Laboratories,
N.V., Pontbeek 61, B-1731 Zellik, Belgium (E-mail:
mailto:penglebienne@redlabs.be ).

SUMMARY. Low molecular weight (LMW) ribonuclease L (RNase L) forms have
been identified in peripheral blood mononuclear celIs (PBMC) of patients
with chronic fatigue immune dysfunction syndrome (CFIDS). Data from our
laboratory indicate that these LMW RNase L proteins are produced by
proteolytic cleavage of the native monomeric enzyme and we have
identified calpain as one of the possible proteases involved. 

Using human
recombinant RNase L (r-hRNase L) His-tagged at the N-terminus, we show
here at the one hand that both calpain and PBMC extracts from CFIDS
patients cleave the protein in fragments of identical sizes containing
ankyrin-like repeat sequences. At the other hand, the activity of RNase L
is modulated by its interaction with a specific inhibitor (RLI), a member
of the ATP binding cassette (ABC) superfamily. RLI interacts with the
ankyrin domain of RNase L, which results in a blockade of the
2',5'-oligoadenylate (2-5A)-binding site of the enzyme. 

We show that RLI
contains a small ankyrin-interacting peptide cluster through which it
interacts with the first two Beta-hairpin coils of the RNase L ankyrin
domain. A similarity search performed at the NCBI using RLI aminoacid
sequence as the entry allowed to identify several other ABC transporter
proteins sharing significant identities with RLI, including the
ankyrin-interacting peptide. Taken together, these results show that upon
pathological cleavage of RNase L, fragments containing the ankyrin domain
are released, which could be capable of interacting with selected members
of the human ABC superfamily, preventing their interaction with the
normal cognate ankyrin protein and hence impairing their proper cellular
function. This interaction constitutes a common physiological mechanism
explaining numerous and currently unexplained symptoms experienced by
patients with CFIDS, which are otherwise totally unrelated.


KEYWORDS. RNase L, ankyrin-like domain, ABC transporters, CFIDS, CFS,
protein interactions, RLI, sequence homology, 3D-models, integral
membrane components, cytoskeleton


INTRODUCTION
Ribonuclease L (RNase L) is an endonuclease central to the 2-5A antiviral
pathway (1). Upon binding small 2',5'-oligoadenylates (2-5A) produced
from ATP by the 2-5A synthetase activated by interferon and double
stranded RNA (ds-RNA), the enzyme homodimerizes. Homodimerization confers
the catalytic activity to the protein which cleaves single stranded RNA
(ss-RNA) (2). The catalytic activity of RNase L is regulated through
interaction with the specific RNase L inhibitor (RLI) (3,4), which
impairs 2-5A-binding to, homodimerization and hence catalytic activation
of the monomeric enzyme. The 2-5A-binding site of RNase L is located in
the ankyrin-like N-terminal domain of the enzyme which is involved in the
interaction with RLI (5).

In chronic fatigue immune dysfunction syndrome (CFIDS), the 2-5A pathway
is dysregulated and peripheral blood mononuclear cell (PBMC) extracts are
characterized by an unregulated RNase L activity, a downregulated RLI,
and the presence of low molecular weight (LMW) forms of RNase L,
including a 37-kDa 2-5A-binding protein (37-kDa RNase L) (6-8). Besides
cellular and humoral immunity dysfunctions (9), patients suffering from
CFIDS present also many symptoms, including pain, which are likely to
reflect dysregulations in both ion and amino acid transport (10,11). The
involvement of ion channel dysfunction in CFIDS has already been
proposed, based on clinical observation (10, 12), as a rationale for some
of the symptoms observed in the illness.

A recent study (13) classifies RLI as a member of the ATP binding
cassette (ABC) superfamily of ion membrane transporters. We show here
that RLI sequence shares strong homologies with other members of the
superfamily, which include a small peptide ankyrin-binding motif
analogous to the tetra- or pentapeptide through which, both the
Na+,K+-ATPase and erythroid anion exchanger AEI interact with the
erythrocyte ankyrin (14,15). We report also that the PBMC of CFIDS
patients contain a proteolytic activity which cleaves RNase L in
fragments containing the ankyrin-like domain. Ankyrins are
heterobifunctional proteins which play fundamental roles in linking the
cytoplasmic domain of integral membrane proteins to the cytoskeleton
(14,15). Our results strongly suggest that the ankyrin fragments released
from RNase L in cells of CFIDS patients are capable of interacting with
members of the ABC superfamily homologous to RLI. This could preclude
their interaction with the normal cognate ankyrin protein and result in a
dysregulation of their normal ion channeling function.


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