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Summer Research Fellowship Programme of India's Science Academies

Probing the dynamic outreaches of Ornithine Urea Cycle: Frameshifting rote perspectives to paint an ulterior picture

Areeba Khan

M.Sc. Biochemistry Sem 3, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025

Dr. Mitali Mukerji

Chief Scientist, Institute of Genomics and Integrative Biology, Mathura Road Campus, Sukhdev Vihar, New Delhi 110025

Abstract

A bizarre interplay, an intricate ramification yet discrete individualism is accosted in the myriads of forms and flares constituting the nature. Biochemical pathways forming the physiochemical basis of such daedal interactions are but probed in a relatively rote stead. Rather, in line with the faculties of Nature; the biochemistry behind bio systems too is a mutually interplayed gross synchrony whereby each pathway ambles hand in hand with the rest while still emblematising selfhood. Under the aegis of this project, we aim to decipher the vast multitude of realms traversed by the pathway pertaining to Urea metabolism and transport while addressing the concept of METABOLOMICS - probing metabolism in light of genomics. Conventionally, this pathway is impregnated into a learner's intellect in a static obsolete frame confined to the principal role of clearance of waste nitrogen in vertebrates, but it's way ahead of this tread. Genes coding for the pathway enzymes and transports offer plethora of dynamic implications from overlapping with diverse pathways, influencing energy metabolism, exerting moonlighting effects to inducing enzymes in rather remote areas, among others. Series of structured modifications have occurred and probation of evolutionary hierarchy can unravel astounding insights into the vast metabolic linkages and developmental intricacies. The pathophysiological implications of Urea Cycle genes on gross system physiology shall unfold the genomic basis of pathological conditions where these genes may play a detrimental role. As such Urea Cycle genes exert vast physiological responses and mutations thus have the potentiality to elicit effects in relatively remote areas. We aim to broaden horizons by interspersing biochemistry into genomics to present an altered perspective for approaching diagnosis and therapeutics. For instance, a cardiovascular problem or even obesity; can have links to an anomaly in urea cycle gene(s). Thus, keeping abreast of biochemical wiring shall be instrumental in unveiling genomic linkages of diseased conditions thereby making treatment of remote causes and not superficial symptoms; accessible. Reading between the lines is the need of the hour and that's precisely what we plan to do.

Keywords: urea-cycle, metabolomics

INTRODUCTION

"There is nothing permanent except change." (Heraticus)

Everything constitutive of this world is in possession of a transient existence. There's a constant evolutionary escapade running in the backdrop of each material as well as living entity. Change is thus an inevitable face of the practical reality we ought to appreciate. Human race is no exception and has also witnessed immense behavioural changes over the course of time. Methodical approach to perception of surroundings, engorging the cognizance, refining the intellect and oozing the output; has undergone a passionate revolution. There's an infinite disparity in the comprehending, learning, memorising and grasping skills of generations bygone and the present youth. Unfortunately, while the world is just at the edge of incurring the Fourth Industrial Revolution; the learning resources as well as their dissemination is still operating on the age old lines. Since I claim a kin to the clan to 21st century robust youth; so I'm quite abreast of the prevalent monotony in dispersal of knowledge. Hailing from a biochemistry backdrop, I'm adept at the conceptualisation of the biochemical pathways operating within biosystems. But much to our dismay; the biochemistry running agile within the entities of the biological world; has been perceived by the developing minds in the traditional rote paradigm of textual information which fails to garner any arousal of the intellect. On face of it, there's an intricate network of varied processes running constantly within each living entity to maintain the state of living. Behind these biological processes; there is a complex interplay of vast biochemical pathways and the genetic foundation of the concerned living system. There's a persistent prevalence of an anachronistic system whereby biochemistry and genetics are pictured before young learners on a parallel track meant to stay aloof and as such the subject in general and the inlaid pathways in particular; are perceived in an unpalatable stance by the blooming minds. Instead; there's no denial to the fact that the biochemistry behind biosystems is but ramified into the genetic backdrop and the two coincide amicably to run the bandwagon of life. Under the aegis of this project, within the auspices of my guide; I explored biochemistry in an altogether different light and comprehended the genomic aspect laid intimately within by opting for Urea Cycle as a model for this mental exercise and while digging deeper into its metabolism and genetics; I could drive upto bizarre potentialities which couldn't otherwise be arived at. Visualising Urea metabolism with its basic genes; as a part of a complex, interwoven and anastomosing network; I came across the broader aspect of metabolism merged within genomics or METABOLOMICS as it can be precisely phrased. I ended up breaking stereotypical paradigms and thereby painting an ulterior picture by frame shifting rote perspectives.

METHODOLOGY

In a quest to perch out of the box and stand out from the shepherd's flock; we employed a meticulous methodology to deal with biochemistry in light of genomics. The idea was to refine mental faculties and redefine biochemical pathways from a genetic foothold. In order to be able to appreciate biological system as a complexly wired network where nothing exists aloof and each process pacing within, is infact coexisting with several others; we used Ornithine Urea Cycle as our model for this mental experiment. We took a coherent and practical approach to deal with the substance at hand and progressed from basics to the core. The first step was to gather ground for the very foundation of Urea Metabolism. As such, the metabolic pathway was traced and it appeared in the same monotonous garb it had always been draped in and henceforth, being the rote pathway illustrated unceremoniously in the conventional learning resources. We then moved a step ahead by incorporating the genomic backdrop of this metabolic pathway and came up with the genes coding for the enzymes and transportes. Next, we explored the diverse metabolic pathways and pathologies linked to Urea Cycle along with exploring the Urea Metabolism genes exerting influences there. Then we traced the vast influences of Urea Cycle genes with respect to pleiotropic, moonlighting etc. to decode their impact on gross system physiology. Having discovered the plethora of attributes impacted by the genes of urea genesis; we finally probed the various diseases of remote as well as vicinity areas, where these genes have been sighted to play a role and comprehended if an up-/down-regulation of the metabolic pathway would exert appreciable influence; in order to arrive at a conclusion. We ended up exploring the explicit potentialities and extremely vast outreaches of the Urea Metabolism genes on system pathophysiology.

RESULTS AND DISCUSSION

During probation of the dynamic outreaches of the Ornithine Urea Cycle while attempting to frameshift rote perspectives to carve an altered perception; we arrived at important realms:

The Branch Offs

Nitric oxide pathway

This metabolic pathway is responsible for nitric oxide (NO) production and shares two enzymes from urea cycle namely Arginosuccinate Lyase (ASL)and Arginosuccinate synthase 1 (ASS1) alongwith a carrier citrin.[1] Oxidation of L-Arginine by molecular Oxygen in the presence of Nitric Oxide Synthase (NOS) releases NO and yields citrulline with the latter being recycled to L-Arginine by ASS1 and ASL. [2] . The endogenous NO plays many significant roles:

1.It regulates ovulation, uterus-oviduct contractility, placental vasculature etc. in female reproductive system and promotes erection of penis, spermatogenesis, steroidal agility etc. in male reproductive system and also, sperm motility is found to vary inversely with NO concentration. [3]

2. It's responsible for vasodialation, angiogenesis, proliferation of myocardium etc. and thus has a crucial role to play in cardiovasculasr system pathophysiology. [4]

3. In the alimentary canal, NO regulates smooth muscle behaviour thereby, affecting important parameters including gastric emptying, intestinal contractions etc. and also seemingly plays a role in release of acid, mucus and alkaline secretions.[5]

4. NO conrols the behaviour of pulmonary vasculature and regulates blood flow parameters such that an imbalance in the NO levels can pose a risk of development of Pulmonary Hypertension.[6]

Thus, NO is an important metabolite and exerts impact on the functioning of a diverse metabolic pathways. The production of NO is intimately associated with Urea Cycle and hence, the concentration of Urea decides the level of NO in the system. As such, any abnormality in the Urea Cycle due to under-/over-expression of the associated genes; shall have detrmental effects on the NO pathway and hence on a wide variety of biosystems associated with the latter thereby exerting vast influence on distant metabolic pathways. Thus, any dysfunction associated with any of the above bio-systems can be traced to an anomalous functioning in the ureagenesis genome.

Tri Carboxylic Acid (TCA) cycle

Breakdown of Arginosuccinate in Urea Cycle releases fumarate that enters TCA and is converted to malate by fumarase enzyme and then to oxaloacetate (OAA) by malate dehydrogenase. [7] Transamination of OAA with glutamate re-forms aspartate that again enters Urea Cycle. [7] CO2 released via TCA cycle is used in the formation of bicarbonate ions that react with NH3 in an energy dependent reaction to generate carbomoyl phosphate of Urea Cycle. [7]Carbomoyl Phosphate Synthase-1 (CPS-1) is activated by N-Acetyl Glucosamine (NAGS) synthesized from Acetyl CoA and glutamate. Thus , Acetyl CoA is shared between TCA and Urea Cycle. [7] Hence., any up-/down-regulation of Urea Cycle due to over-/under-expression of gene(s) shall lay substantial impact on TCA cycle and therefore on energy metabolism while the vice-versa also equally holds good.

Gluconeogenesis

Gluconeogenesis and Urea Cycle are intimately associated such that the two pathways share certain enzymes including malate dehydrogenase, aspartate aminotransferase, alongwith transporters of malate, glutamate, aspartate and α-ketoglutarate. [8] A precursor from gluconeogenesis (pyruvate or lactate) dictates ureagenesis from NH3 but elevated levels of NH3 can abort gluconeogenesis. [8] Thus, a tight regulation of genes responsible for urea synthesis and gluconeogenesis is required to operate gluconeogenesis as well as urea cycle in liver such that any anomaly shall impact the metabolic pathways and hence glucose and urea concentation as well as energy saturation inviting a host of pathophysiological implications.

This aspect of metabolism is often eclipsed in the traditional method of teaching while it's absolutely important to appreciate the metabolic pathways as interconnected webs rather than discrete pockets so that a wide horizon can be understood whereby genomic imbalance in a particular pathway might inluence a remote pathway owing to the intimate metabolic linkages.

Acid Base Balance

Bicarbonate ions are irreversibly removed via urea cycle in liver (carbonyl group in urea comes from bicarbonate ions) in a reaction sensitive to the extracellular pH status and thus urea cycle seemingly plays a role in maintaining acid-base balance of the body suggestive of an operational feedback loop. [9] ​​​​

Diversified Pathological Conditions Influenced by Urea Cycle Genes

Diseases influenced by Urea Cycle genes ​
 DISEASEGENE(s) INVOLVED 
 Acidoses CPS-1, OTC
 Acute Necrotizing PancreatitisARG-I 
 Acute phase reactionASS1 
 Alzheimer's diseaseASS1 
 Fetal growth retardationARG1, CPS1, OTC 
 Hepatocellular carcinomaARG1, ASS1 
 HypercapniaARG1 
 HyperoxiaARG1, ARG2, CPS1 
 HypoxiaASL, ASS1 
 Immediate hypersensitivityARG1, ARG2 
 Insulin resistanceARG1 
 Liver failureARG1, ASL, ASS1, CPS1, OTC 
 Lung cancerCPS1 
 Musclar diseasesARG1 
 Neonatal pulmonary hypertensionARG2 
 ObesityCPS1, OTC 
 Optic nerve injuriesARG2 
 Pulmonary hypertensionARG2, CPS1 
 Retinal degenerationARG2 
 SepsisOTC, ARG2 
 Syndromic X lnked intellectual disability Lubs typeOTC 
 Trichohinophalangeal syndrome type 1CPS1 
 Type 2 diabetes mellitusASL 
 Veno occlusive diseaseCPS1 
Note
The table above highligthts some of the vast multitude of diseased conditions in rather distant areas where urea cycle genes have been sighted to play a role..​​​

Vast Pathways Influenced by Urea Cycle Genes

Urea cycle genes in varied metabolic pathways ​​
 PATHWAYGENE SYMBOL(S) 
 2-hydroxyglutaric aciduria pathwayCPS1
 AGAT deficiency pathwayARG1, ASS1, ASL, CPS1, OTC 
 Alanine, aspartate and glutamate metabolic pathwayASL, ASS1, CPS1 
 Arginine and proline metabolic pathwayARG1, ARG2, ASL, ASS1, CPS1 
 Canavan disease pathwayASL, ASS1 
 Entamoebiasis pathwayARG1, ARG2 
 Glutamate metabolic pathwayCPS1 
 Homocarnosinosis pathwayCPS1 
 Hyperprolinemia type 1 and 2 pathwayARG1, ASS1, ASL, CPS1, OTC 
 Interleukin-4 signalling pathwayARG1 
Succinic semialdehyde defiiency pathwayCPS-1 
Note
Table 2 above illustrates the participation of the urea metabolism genes in the system physiology by influencing various metabolic pathways.​

Important Mention

  • CPS1 has been found to be associated with the risk of development of Coronary Artery Disease (CAD) in a gender specified manner such that it is responsible for low low titre of urea metabolites and high blood glycine repertoire thereby standing for low risk of CAD in females. [10]
  • Single nucleotide polymorphisms (SNP) in CPS1 have been sighted to be responsible for development of Pulmonary Hypertension in Newborns (PHN) because mutations in CPS1 affect urea metabolism and thereby the associated NO pathway hence, resulting in PHN. [11]
  • Mitochondrial enzyme CPS1 catalyzes the rate determining step of ureagenesis converting NH3 (derived from glycine) and bicarbonate ions (derived from metabolic activity) to Carbomoyl Phosphate such that CPS1 and glycine are linked via the folate pathway and urea cycle and association of glycine to the insulin responsiveness as well as to the the metabolism of osmolyte betaine; is suggestive of a role of CPS1 and hence, Urea Cycle in controlling body weigth post weightloss. [12]
  • rs278166 SNP in ARG1, enhances NO-based apoptotic action in lymphocytes and thus, lowers the risk of encounter of Pulmonary Hypertension in sufferers of Bronchopulmonary Dysplasia. [13]
  • A potential role of Urea Cycle has been sighted in the patho-physiology of Alzheimer's Disease (AD) such that indivisuals at risk of AD have significantly raised expression of OTC in their brains. [14]
  • SNP at rs5963409 in OTC results in hypertension as well as elevated vasoconstriction activity giving air to a probable involvement of urea pathway in regulation of blood pressure. [15]
  • The innate type 2 lymphoid cells alongside the episode of Type 2 inlammatory responses are mediated by ARG1 in a cell intrinsic mannerism thereby offering newer potentialities in the approaches towards treatment of inflammatory diseases amongst human. [16]
  • Arginine and Tryptophan metabolism is intricately interwoven such that Indoleamine 2,3 dioxygenase 1 activity is dependent upon ARG1 expression and so is the dendritic cell signalling activity of the former thereby, making ARG1 an important contender of immuno-metabolism. [17]
  • Upregulated ARG-1 activity in vascular endothelium, lowers L-Arginine repertoire thereby resulting in reduced NO levels and elevated oxidative stress which invites cardiovascular problems post intake of fat rich diet. [18]
  • Arginase is'nt just confined to urea metabolism and is known to exert moonlighting effects in diverse realms including modulatory action on host immune machinery upon microbial infestation though the inlaid mechanisms still need deeper probation. [19]
  • Denaturation of Calcium/Calmodulin dependent protein kinase IV - a multifaceted enzyme which is responsible for monitoring memory, genetic expression, bone development, cell cycle, T-cell differentiation etc.; is induced by Urea in humans [20] and as such Urea Cycle is therefore likely to lay impact on the above mentioned diversified and crucial metabolic activities.

Thus, Urea Cycle genes cease to remain confined to urea metabolism and instead have ample of impacts on distant pathways and disease pathophysiologies which needs to be appriciated in the present teaching methodologies to bring out the true essence of the concept of METABOLOMICS.

CONCLUSION AND RECOMMENDATIONS

The lithe journey of this project has been a bizzare one thereby ornating the concepts of metabolism and genetics in an altogether distinct flare. My heart had skipped a beat when my guide had proposed a probation of metabolism and genomics of urea cycle as my project substance because I had always been under the the rote, unamusing and unpalatable impression of the pathway confining it to only urea synthesis like it has always been disseminated in the traditional textbooks. But when I embarked this journey; I explored urea metabolism like I had never imagined even in the vast wildernesses of my thoughts. I could realise the conceptualisation of METABOLOBICS and was able to link the genes pertaining to urea metabolism with the physiology of the entire system as a whole and arrived at the conclusion that Urea Cycle genome (CPS1, ARG 1&2, OTC to a greater extent and ASS1 & ASL to a slight lesser, though highly appricable extent) asserts a detrimental role on the gross system pathophysiogy. This dictates the importance of thoroughly investigating the genomic basis of diseased conditions with respect to the metabolic webs in order to catch hold of a remote cause and not remain confined to a superficial symptom so as to give wider and refined realms to the dynamics of diagnostic and therapeutic methodologies. I would recommend the readers to rise above the anachronistic paradigms and become appriciative of exploring bio-systems as intimately anastomosed networks to bring to light astounding potentialities waiting to be explored in the web of metabolism within the cores of genomics. The auspices of this project lifted off my monotonous harp on the same string and helped me frameshift my perspectives to paint an ulteroir picture of biochemistry in light of genomics.

ACKNOWLEDGEMENTS

I would like to extend my warmest gratitude to Allah for caliberating me capable enough to plunge into this venture. I hereby sincerely acknowledge the INSA-IAS SRF conducting body for their excellent practice of helping young budding scientists like me, to get the much needed research exposure and refine the scientific calibre during the extended summer break. I also extend a sincere vote of thanks to my reverend guide Dr. Mitali Mukerji who made me realize my potentialities to the extreme and come up with the best results. Equally important to me is the constant support of my parents and Yasar during the course of this project. This project is dedicated to all those who helped me somehow during the journey.

" There's nothing permanent except CHANGE."

- HERATICUS

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Source

  • Table 1: https://rgd.mcw.edu/rgdweb/pathway/PathwayRecord.html?acc-id=PW:0000076&species=Human#annot
  • Table 2: http://rgd.mcw.edu/rgdweb/pathway/pathwayRecord.html?acc-id=PW:0000076&species=Human#annot
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