Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

Abstract

Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised.

An energetic view of stress: Focus on mitochondria

“…Interestingly, the biosynthetic enzymes involved in catecholamine degradation, the monoamine oxidases MAO-A and MAO-B are anchored to the outer mitochondrial membrane (Binda et al, 2011). Some reports also suggest that TH may be tethered to the mitochondrial surface (Wang et al, 2009; Baumann et al, 2016), although this may be tissue specific and inducible under certain conditions; more work is needed to examine this potential functional association of mitochondria with catecholamine synthesis and degradation.…”

Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

“…The interaction of proteins with cellular membranes controls fundamental cellular processes such as membrane trafficking, cytokinesis, and intracellular signaling [1][2][3][4][5][6][7]. Beside cytoplasmic, peripheral or integral membrane proteins, there is a class of proteins known as amphitropic that interacts with lipids and membranes during their biological activity [8,9].…”

“…Proteins may also affect membrane integrity leading to cellular dysfunction or death [2,[14][15][16]. This is the case for the interaction between partially unfolded proteins/protein aggregates and lipid bilayers [17][18][19][20][21] possibly generating cytotoxicity and, in vivo, the onset and progression of several human diseases [2,4,15,16,19].…”

“…Proteins may also affect membrane integrity leading to cellular dysfunction or death [2,[14][15][16]. This is the case for the interaction between partially unfolded proteins/protein aggregates and lipid bilayers [17][18][19][20][21] possibly generating cytotoxicity and, in vivo, the onset and progression of several human diseases [2,4,15,16,19]. Membraneprotein interactions are of the same origin [19,22,23] and are regulated by electrostatics, hydrophobic forces [24,25] and protein flexibility [26], being this true for both pathological and non-pathological proteins [15,[27][28][29].…”

A Perspective: Active Role of Lipids in Neurotransmitter Dynamics

“…Dopamine, the first catechol NT at the biosynthetic pathway, is synthesized from the amino acid tyrosine in two steps: (1) the tyrosine hydroxylase converts it to L-dopa, and (2) the L-dopa decarboxylase converts it into dopamine. Although both enzymes were previously considered to be cytosolic, recent studies have indicated their association with membranes: the tyrosine hydroxylase binds to liposomes [123], and the L-dopa decarboxylase associates generally with the membranes in the cellular environment [124]. In the next step, dopamine is used as a substrate in the synthesis of two other catechol NTs: (1) norepinephrine and (2) epinephrine.…”