Augmenter of Liver Regeneration: A Fundamental Life Protein

ALR and Liver Regeneration

Initial ALR studies showed hepatic protection against injury associated with portal vein diversion.⁽⁵⁾ ALR stimulates primary human hepatocytes in vitro with a potency comparable to that of hepatocyte growth factor and up-regulates several enzymes critical for polyamine synthesis, an essential element of early liver regeneration.⁽⁶⁾

ALR is released immediately following partial hepatectomy. This protein activates the mitogen-activated protein kinase pathway, inhibits lytic activity of natural killer cells, and up-regulates interleukin-6 and tumor necrosis factor-α as well as inducible nitric oxide synthase production by Kupffer cells, the latter leading to release of nitric oxide.⁽⁷⁾ Short ALR protects hepatocytes from apoptosis induced by various stimuli,⁽⁸⁾ and reduction of ALR in the partial hepatectomy model has deleterious effects on liver regeneration.

ALR and Mitochondrial Protein Import

The mitochondrial intermembrane space (IMS) is the conduit by which cytoplasmic proteins access the mitochondrial matrix. The disulfide relay system, also called the mitochondrial intermembrane assembly (MIA) system, consists of two proteins, MIA40 and long ALR, that mediate oxidative folding and entrapment of proteins destined for IMS localization. The name “MIA system” implies that MIA40 is the sole critical element, but it is a misnomer as ALR is responsible for the IMS localization of MIA40⁽⁹⁾ and for the continued reoxidation that permits MIA40 to function. ALR subsequently transfers electrons through its attached flavin adenine dinucleotide (FAD) cofactor to cytochrome c and thence to oxygen as the final electron acceptor. This combined enzymatic and electron transfer capability has led to the description of ALR and related proteins as “a unique and remarkable new FAD [flavin adenine dinucleotide] binding fold family.”⁽¹⁰⁾ ALR serves as a link between MIA protein import and energy production by the electron transport system. This not only increases protein import efficiency and avoids oxygen radical formation but also places oxygen in a position to affect the process. Indeed, the disulfide relay system, in particular ALR, has been suggested to function as a cellular oxygen sensor, up-regulating MIA40 redox activity in the setting of high oxygen levels and down-regulating it in the opposite circumstance.⁽¹¹⁾ Multiple proteins affected by this process participate in detoxification of reactive oxygen species, contribute directly to respiratory chain or adenosine triphosphatase complexes,⁽¹²⁾ and protect the mitochondrial genome from oxidative stress.⁽¹³⁾

Additional features point to ALR as the central protein of the disulfide relay system: evolutionary divergence in Trypanosoma brucei shows that ALR alone is able to mediate IMS protein folding in the absence of Mia40,⁽¹⁴⁾ and ALR, but not MIA40, responds to alterations of mitochondrial DNA levels or mitochondrial DNA mutation.⁽¹⁵⁾ This second point suggests that ALR could represent a regulatory molecule in this pathway.

ALR, Oxidative Injury, and Apoptosis

Mitochondrial ALR depletion increases oxidative stress, causes rapid adenosine triphosphate reduction, and causes cell death by apoptosis and necrosis.⁽¹⁶⁾ Conversely, ALR inhibits apoptosis in activated lymphocytes,⁽¹⁷⁾ renal proximal tubular cells, neuroblastoma cells, glioma, and skeletal muscle cells.⁽¹⁸⁾

Analysis of primary hepatocytes showed protection against apoptosis from a variety of stimuli.⁽⁸⁾ Separate studies⁽¹⁹⁾ show that the antiapoptotic effect of ALR is mediated by immediate facilitation of antioxidant accumulation within mitochondria as well as by increased Bcl-2 and secretory (antiapoptotic) clusterin. In summary, ALR exhibits strong cellular protection and prosurvival functions.

ALR and Iron-Sulfur Proteins

Mitochondria are the site of iron-sulfur cluster assembly with a separate export machinery to deliver these proteins to the cytoplasm and nucleus. Integral to this export machinery is ALR.⁽²⁰⁾

ALR was shown to be responsible for Fe-S cluster transfer and stability of the mitochondrial outer membrane protein mitoNEET which repairs oxidative injury to aconitase/iron regulatory protein-1, a coordinator of genes mediating iron homeostasis and oxygen sensing.⁽²¹⁾ Defects of both ALR/Erv1 and MIA40 lead to intramitochondrial iron accumulation.⁽²²⁾

The use of Fe-S compounds to harness energy by primitive life-forms in hydrothermal vents reflects early bioenergetics preceding an oxygen-rich atmosphere.⁽²³⁾ Eukaryotic cells can withstand anoxia given sufficient fermentable carbon, but they cannot survive in the absence of Fe-S protein biogenesis. Fe-S proteins function in protein translation and ribosome maturation, likely reflecting the transition from catalysis in an Fe-S–containing world to ribosomal protein synthesis in cells.⁽²⁰⁾ The participation of ALR in this process strengthens its status as an ancient protein operating at the level of fundamental life processes.

ALR and Intermediary Metabolism

Absence of hepatic ALR leads to profound steatosis with elevations in triglyceride and cholesterol levels⁽²⁴⁾ due in part to reductions of several enzymes important for fatty acid β-oxidation, including the mitochondrial transport protein carnitine palmitoyltransferase-1a and peroxisome proliferator–activated receptor-α, which regulates the fed/fasted state and controls a gene network involved in using fat as an energy source.

Loss of ALR also leads to reduced sterol regulatory element binding protein-1c, up-regulating sterol regulatory element binding protein-2 transcription, which in turn stimulates genes involved in cholesterol synthesis.⁽²⁵⁾ This, in addition to increased acetyl-coenzyme A related to decreased fat catabolism, likely contributes to increased cholesterol levels. In short, current findings place ALR at the crossroads of hepatic lipid and cholesterol metabolism.

This activity of ALR is not dependent upon its sulf-hydryl oxidase. Administration of short ALR reduced free fatty acid–induced hepatocyte lipid accumulation in murine cells⁽²⁶⁾ and was associated with alterations in microRNA 122, indicating a separate mechanism.

These pleiotropic effects of ALR, combined with its constitutive mitochondrial activities linked to energy transduction and oxygen sensing, suggest a role as a critical component of a “hepatostat” that maintains the liver as the central regulator of metabolic and energy homeostasis.

ALR and Stem Cells

ALR is up-regulated in embryonic and adult stem cells⁽²⁷⁾ and plays a significant role in maintaining “stemness.”

Nuclear ALR opposes c-Jun activation domain– binding protein-1–mediated nuclear export of the cell cycle inhibitor p27kip1, thus maintaining an interphase stem cell population.⁽²⁸⁾ ALR plays a separate role in embryonic stem cells. Here, mitochondrial ALR down-regulates dynamin related protein-1–mediated mitochondrial fission, maintaining mitochondria in a primitive state and maintaining stem cell pluripotency.⁽²⁹⁾ ALR knockdown allows escape from this state. At present, manipulation of ALR has been harnessed to stabilize and maintain pluripotency of therapeutic embryonic stem cells.⁽²⁹⁾

These observations also imply a role for ALR in early development. ALR expressed in zebrafish played a major role in liver outgrowth through stimulation of hepatocyte proliferation.⁽³⁰⁾ ALR knockdown inhibited not only liver growth but also that of exocrine pancreas and heart,⁽³¹⁾ indicating a role in these organs as well.

ALR and Disease

Conclusions

ALR is one of the ancient iron-sulfur proteins linked to the earliest means by which life first channeled environmental energy. Cellular energy production is thought to be even more fundamental than DNA in driving cellular complexity.⁽⁴⁴⁾ This essay has touched upon the myriad roles of ALR in selected basic life processes. Additional roles in spermatogenesis, where it is expressed at highest levels,⁽⁴⁵⁾ or in the CNS, in which it has been mapped to all parts of the brain⁽⁴⁶⁾ but remains unstudied, represent further areas of investigation. New tools such as a specific ALR inhibitor⁽³¹⁾ may lead to more focused studies in the near future. Far from being limited to a significant role in liver regeneration, ALR is essential for respiration and growth in all eukaryotes, from yeast to man.