ECTO-NOX PROTEINS

ECTO-NOX proteins are a family of cell surface proteins with common characteristics. The ECTO-NOX proteins are included in the HUGO Human Gene Nomenclature Data Base genenames.org and are designated as ENOX proteins to distinguish them from other NAD(P)H oxidase including the PHOX-NOX proteins of host defense located at the cylosolic suface of the plasma membrane. CNOX is ENOX1 or ECTO-NOX disulfide-thiol exchanger 1 (EntrezGene ID: 55068). The tumor-associated tNOX is designated ENOX2 or ECTO-NOX disulfide-thiol exchanger 2 (EntrezGene ID: 10495). The age-related or arNOX proteins constitute a third broad category of ENOX proteins. These arNOX proteins generate superoxide and appear related to the aging process.

ECTO-NOX or ENOX proteins are reduced pyridine nucleotide oxidases that differ from all other reduced pyridine nucleotide oxidase in an absence of flavin and resistance to inhibition by cyanide. Except for ECTO-NOX proteins all other reduced pyridine nucleotide [NAD(P)H] oxidases require bound flavins for activity, their activity is inhibited by cyanide or requires flavin or both.

ENOX proteins exhibit a second catalytic activity referred to as protein disulfide-thiol interchange. A characteristic C (for the amino acid cysteine)-X (any amino acid)-X-C motif is common to most, if not all, members of the protein disulfide isomerase family of proteins and present, as well, in thioredoxin reductase and related proteins. The motif appears to catalyze the transfer of electrons in conjunction with bound flavin making them unique in their molecular make up chromosomal origins. Both the CXXC motif and bound flavin are lacking in ENOX proteins.

The physiological substrate for the oxidative activities of the ECTO-NOX proteins appears to be reduced coenzyme Q10 of the plasma membrane rather than NADH directly. The proteins will reduce NADH but the NADH binding site is on the outside of the cell where there normally is little or no NADH. Rather, NADH inside the cell is used to reduce the plasma membrane quinones at the inside surface of the cell membrane and the reduced quinones move across the membrane to the outside where the reduced quinone coenzyme Q10 is then oxidized completing the transfer of protons and electrons coming from NADH to some external acceptor (normally molecular oxygen).

The two activities catalyzed by ECTO-NOX proteins, the oxidation of NADH or reduced coenzyme Q10 and protein disulfide-thiol interchange, do not occur continuously. The activities pulse with a 2 + 3 pattern (two pulses more widely separated than the other three) that generates a very precise period length that is characteristic for each family member. The two activities alternate with the oxidative activities coming on the two more widely separated maxima and the protein disulfide-thiol interchange activities coming on the 3 less widely separated maxima.

For the most part ECTO-NOX proteins in their natural environment are resistant to degradation (including N-terminal amino acid sequencing and protease or chemical digestion). As a result they are extremely stable.
Except for tNOX and arNOX, there are no known inhibitors of ECTO-NOX proteins. This together with their extreme stability means that ECTO-NOX proteins can withstand and continue to function even under the most adverse conditions within a cell or organism.

ECTO-NOX proteins have properties of prions (stability, protease resistance, and the ability to learn and teach — i.e. to adopt different changes in structure and then transmit these changes to like molecules so that an entire population ends up with the changed characteristics) which may be related to the property of chemosensitization of tNOX proteins to cancer therapeutic drugs.

Biological function of ECTO-NOX proteins

To the extent that the process is understood, the principal biological function of ECTO-NOX proteins in general is as terminal enzymes of plasma membrane electron transport. In this process, proton (H+) and electrons (e-) are carried from point A (the inside of the cell) to point B (the outside of the cell) and donated to some appropriate acceptor such as molecular oxygen. The inside-of-the-cell donor is NADH which becomes oxidized to NAD+. Coenzyme Q (Q) acts as a shuttle to transport the protons and electrons across the cell membrane by itself becoming reduced and reoxidized. The oxidation of the reduced Q is carried out by the cell surface ECTO-NOX proteins. This function in a modified form may explain how prions which exhibit ECTO-NOX activities and beta-peptides of Alzheimer’s disease which also exhibit ECTO-NOX activities function to kill nerve cells. They attach to the nerve cell membrane, form channels and drive protons and electrons inside which may also contribute to toxic excess entry of ions such as calcium.

For tNOX and CNOX a second biological function is the essential role of the protein disulfide-thiol interchange activity in growth.

The part of growth that requires ECTO-NOX function is cell enlargement. When a cell divides to form two progeny cells, the cells have about ½ the volume of the original precursor cell. In order to divide again, the progeny cells must increase their volume (enlarge) to reach a size very near to that of the original precursor cell. It is this process that is driven by ECTO-NOX catalyzed protein disulfide-thiol interchange.

Finally, it is clear that ECTO-NOX proteins function in the cell enlargement phase of cell growth not as individual protein molecules but as part of a complex. We have reconstituted such a complex in a completely cell-free system that uses lipid vesicles and recombinant proteins. The minimal requirements for the functional complex are the ECTO-NOX protein, the associated AAA-ATPase which is postulated to actually drive cell elongation and a source of membrane protein to provide thiol groups. We are able to fulfill the latter function by using a single protein source of little or no specificity such as partially reduced serum albumin.

In normal cells the cell enlargement function is carried out by CNOX. In tumor cells, tNOX dominates in this function even though CNOX may be present. If tNOX is inhibited, the cells cannot enlarge following division and are prevented from dividing again. Normally, the small cells, prevented from enlarging and prevented from dividing eventually undergo programmed cell death (apoptosis). This is the basic therapeutic strategy that underlies the use of tNOX as a target for cancer-specific antitumor agents.

A third function of ECTO-NOX proteins, primarily restricted to CNOX, is that of the ultridian (period length less than 24 h) driver of the biological clock that may ultimately maintain organisms on a 24 hr (circadian) day. The time-keeping role derives from the unique features of ECTO-NOX proteins in general and CNOX specifically in that the two enzymatic activities they catalyze, hydroquinone (NADH) oxidation and disulfide-thiol interchange, alternate within a 24 min period. The ECTO-NOX proteins carry out hydroquinone (NADH) oxidation for 12 min and then that activity rests. While the hydroquinone (NADH) oxidative activity rests, the protein engages in disulfide thiol interchange activity for 12 min. That activity then rests as the cycle repeats. The alternation of activities imparts a time-keeping function to the protein. The length of the period is temperature independent and entrainable, both features of the biological clock. Site directed mutagenesis (cysteine to alanine replacements) of the tNOX cDNA has generated tNOX forms with period lengths of less than and greater than 24 min. COS cells were transfected with a tNOX protein with period lengths longer than 24 min, i.e., 36 or 40 min exhibited a circadian period of 36 h or 40 h in addition to the normal 24 h period length. A 22 min ECTO-NOX period generated a 22 h circadian day also observed in cancer patients. Therefore, we conclude that the circadian period length is 60 X the ECTO-NOX period and that one function of the NOX proteins is to serve as pacemakers for the normally 24 h biological clock.
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