Ulrich Hammerling, PhD

Lymphocyte homeostasis, the balance between cell generation, survival and cell death, represents a tightly controlled process that involves multiple immunologic factors. Like most other cells, lymphocytes are also subject to regulation by general cell biological principles. Previous research in our laboratory has added vitamin A to the list of essential regulators of lymphocyte survival. On all accounts, regulation is effected by vitamin A itself through a cytoplasmic signal cascade, and to a much lesser extent through retinoic acid via transcription.  Direct participation of retinol in cell regulation represents a new branch of vitamin A physiology.  Our findings are relevant to several incompletely explained manifestations of the nutritional vitamin A deficiency syndrome, notably immunodeficiency, still a major health problem. To elucidate the underlying cell biological and biochemical reasons remains one of our main goals.

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While searching for the primary target molecules of vitamin action we discovered that all members of the protein kinase C and cRaf families tightly bound vitamin A at the conserved cysteine-rich domains. Binding, however, had no effect on function as induced by classical hormone-receptor signaling. By contrast, the alternate activation pathway by reactive oxygen species required vitamin A as cofactor.  The current goal is to uncover the mechanism by which oxidative stress and vitamin A cooperate to activate PKC and cRaf. We postulate that oxidation is targeted to conserved vitamin A binding subdomains: these are without exception zinc-coordinated folds that are based on the spatial arrangement of  3 cysteines and one histidine amino acid at the corners of a tetrahedron with a zinc ion in its center. A composite of two such basic building blocks form the zinc-finger domains proper. The latter can be regarded as the nerve center of serine/threonine kinases as they contain the sites where di-acyl-glycerol or GTP/ras bind and activate PKC or Raf, respectively. We have observed that oxidation leads to disulfide formation and relocation of zinc ions. Based on these findings we have formulated the working hypothesis that redox activation of PKC and cRaf by the directed oxidation of special zinc-finger cysteines, catalyzed by vitamin A bound nearby, leads to disassembly of the zinc-finger and consequently to major conformation change. Retraction of the regulatory from the catalytic domain as a means of releasing autoinhibition is the prevailing theory to explain PKC and Raf activation. Our experimental results concurred and showed that redox activation recapitulated many, if not all, of the sequelae initiated by classic pathways. For instance, the redox-activated PKC theta isoform of T cells, like their phorbol-ester activated counterpart, converted to a membrane-bound form and homed to lipid rafts.

Beyond the new insight into mechanisms of kinase activation, the functioning of PKC zinc-finger structures as flexible hinges, just like their bacterial ortholog of the Hsp33 chaperone, challenge the view that similar structures present in a profusion of proteins, from transcription factors, DNA repair enzymes, to ubiquitin ligases, merely serve as rigid clamps. They suggest a general dynamic way by which chemical signals can be translated into changed protein conformation and altered action.

Our second goal is to understand the cell biological reasons of why nutritional vitamin A deficiency engenders immune dysfunction.  This deficiency syndrome is reflected in the marked sensitivity of cultured B and T lymphocytes to vitamin A withdrawal, leading rapidly to mixed necrotic/apoptotic cell death. Multiple cellular systems suffer damage reflecting probably the multiplicity of target molecules interacting with vitamin A, that is: close to a dozen PKC isoforms and three Raf isoforms. Mitochondria, however, stand out in that presence of physiological amounts of vitamin A is critical for their integrity. Vitamin A depletion leads to depolarization of the mitochondria membrane and consequently to loss of ATP production by oxidative phosphorylation. While cytochrome C release initiates the caspase cascade and induces apoptosis in a minority of cells, the rapid loss of ATP shunts the majority of lymphocytes to the necrotic death pathway. How vitamin A is utilized in the regulation of mitochondria has emerged as a pivotal research question. The solution is likely to shed light on the deeper biological question of why nature has chosen to make higher organisms dependent on nutritional sources rather than allowing autonomy through biosynthesis of vitamin A.

[Enlarge Image] Heuristic schematic diagram depicting the action of retinoids in redox activation of PKCa

The PKCa molecule is shown in its accepted domain structure (from "N" to "C" terminus: pseudosubstrate domain as yellow circle; cysteine-rich domains C1A and C1B in magenta; the C2 and catalytic domains in plum).

Reactive oxygen is proposed to oxidize cysteines in cysteine-rich domains, compromising the ability to chelate zinc and causing the zinc-finger to open (X and box-like symbols in green represent intact and oxidation-compromised zinc-coordination centers, respectively).