The Student to Stemette programme has been the best experience last year had to offer. Among the external chaos of working through a pandemic and starting university, being a Stemette Sherpee brought a great sense of internal balance and focus as I sharpened my professional skills and put them to the test under the guidance of an inspiring woman in STEM.
My first step into the STS programme was the matching event in late July – a taste of speed-networking before the mentorship even began! Speaking to several Sherpas (STS mentors), I gained confidence in presenting myself and my aspirations in under a minute and I learnt about an array of exciting and diverse career options in STEM. Best of all, I met Rachel Harris, a management consultant who I instantly clicked with; it turned out, we had both gone to The Tiffin Girls’ School and immediately reminisced about being taught by a few of the same teachers who have been at the school for decades. At the time, I had only been an alumna for a few weeks, and it was incredible to be mentored by another alumna; in Rachel, I found a surprising, final connection to my high school and someone who understood its wonderful traditions, academic pressures and the lasting mindset it leaves you.
Our first mentoring session was mainly delighting in the fact that we had so much in common – from our interest in programming to our love of cats to our many ongoing crochet projects! I also learnt how to deliver an elevator pitch, advertising myself, without hesitating or using any filler words – a tricky task made possible with Rachel’s advice and encouragement.
Next we moved on to SMART goal setting so that I could make the most out of this mentorship. I used to dread making to-do lists but learning to keep my goals Specific, Measurable, Achievable, Relevant, and Time-bound has helped me feel more in control and proactive, not just in the STS programme but in my other university coursework projects too. As part of the mentorship, my aims for the next few months were to:
Secure an 8-week long, project-based internship at a lab at a biochemical firm or research group by May 2021. Work with Rachel to apply for internships in the December 2020 holidays.
Learn how to use LinkedIn effectively as a student with Rachel’s help and through reading articles on making the most of LinkedIn. Aim to build between 20 and 40 connections by Jan 2021
CV/Cover Letter
Update CV and learn how to write an eye-catching CV during the December 2020 holidays
Write a cover letter as part of an internship application by February 2021
Set up a good organisation system for notes and essays and start consistently meeting deadlines by 20 November 2021. Establish a good study/life balance and attend 3 meet and greet sessions / society events and practice networking skills
We tackled the most pressing goal first by discussing how to network, both in formal events and in more casual social settings. The key to effective networking, I learnt, is to be positive, enthusiastic, and authentic and to keep a goal in mind when forming connections with new people. There are many different styles of networker, from idea sharers and specialists (who have a deep knowledge of their subject) to connectors (who proactively form connections for others), and both deep and broad networks. I realised I had a small but deep professional network and now I needed to branch out. Stemettes supported me in this endeavour by organising networking events with other Sherpees; a wonderful series of evenings getting to know my peers on the programme, discussing our experiences with mentorship and bonding over our shared love of science and tech!
Inspired by the positive networking experiences I had within the Stemettes community, I decided it was time to push myself a little further out of my comfort zone so I signed up to an EmpowHER virtual mixer – a chance to meet women in STEM at all stages of their careers. I had an amazing time and made some great connections which I strengthened over LinkedIn! Most importantly, I connected with Stephanie Hay, a consultant who is advising for the neuro-oncological company CancerMune. I delivered a little elevator pitch about myself and told her that I’d be delighted to intern with the company and, to my utter surprise and delight, she arranged a zoom call for the following week to discuss my goals and the possibility of an internship!
I was simultaneously ecstatic and terrified. Prepping for an interview without any guidance in the past had always felt a bit like trying to write a powerful manifesto in a foreign language equipped with only a pocket dictionary. I was keenly aware that I lacked the skills to present myself to employers and this shattered any facade of confidence I could muster. But with the big day looming ever closer, I decided to admit this to Rachel. In just an hour, she taught me how to present myself honestly and authentically, how to demonstrate my knowledge and experience and how to respond to tricky questions that trip up even the most capable of candidates. I ended our Teams call feeling a little less nervous and a lot more excited at the prospect of an interview.
My interview prep with Rachel had paid off; despite shaking like a leaf through most of it, I had managed to make a great impression on Stephanie who invited me back for a second interview with the CEO of CancerMune, Oscar Vazquez. Armed with the confidence of prior interview success, I had a fantastic discussion about glioblastoma treatment and bioinformatics – culminating in a summer internship offer!
In our most recent session, Rachel and I reviewed the goals we set many months ago. Life threw me a few curveballs and surprise successes that meant we didn’t follow this plan to the letter, however setting goals helped us stay on track and proved to me how far I had come in just a few months. I’d managed to attend my first networking event, my first interview, and secure my first proper internship all in the span of a few months – something I could have never dreamed would be possible! I owe so much of it to the STS programme and to Rachel, for her incredible encouragement and advice and I strongly urge any young woman just starting out in STEM to join the Stemettes Society and the STS programme.
The following is a tutorial essay written for the Cellular Biochemistry module – the writing style is pitched at university-level but can also be followed by A Level biology students.
If you’re curious about the intricacies of glucose synthesis in the liver or just want to see what an Oxford biochemical tutorial essay looks like – read on!
The glucose-alanine cycle, also known as the Cahill cycle, describes the metabolic pathway in which extrahepatic tissue (notably, the skeletal muscle) exports alanine to the liver, alanine is used as a substrate in gluconeogenesis, and nascent glucose is exported back from the liver to the other tissues.
Alanine (an amino acid) from broken down protein in our muscles is transported to the liver to be converted into glucose (gluconeogenesis) and recirculated to the muscles to fuel exercise
The gluconeogenic pathway of the Cahill Cycle with irreversible, regulated steps highlighted
Cortisol, glucagon, and insulin regulate the gluconeogenesis pathway
ALANINE –> PYRUVATE
Under extreme catabolic conditions, such as fasting, intracellular protein hydrolysis rate exceeds the rate of its resynthesis – resulting in the liberation of free amino acids which can either be oxidised for energy (in the case of the branched chain amino acids, leucine, isoleucine, and valine) or transported out of skeletal muscle into the bloodstream for gluconeogenesis in the liver.
In the liver, alanine is first deaminated in a transamination reaction, catalysed by hepatic alanine aminotransferase, in which α-ketoglutarate acts as an amino group acceptor. Overall, this reaction is
Alanine + α-Ketoglutarate ⇄ Glutamate + Pyruvate.
Aminotransferases are cytosolic enzymes, particularly abundant in liver cells, which require the coenzyme pyridoxal phosphate (PLP) to be bound to the active site. Transamination of alanine produces pyruvate and glutamate. Glutamate can then be deaminated by glutamate dehydrogenase to reform α-ketoglutarate which can enter the Krebs cycle; this anaplerotic reaction also forms NH4+ which enters the urea cycle to be safely excreted.
PYRUVATE –> OXALOACETATE
The pyruvate can leave the Cahill cycle at this point and be oxidised for ATP production; in periods of fasting, however, it is energetically favourable to remain in the cycle. The conversion of pyruvate into oxaloacetate, catalysed by pyruvate carboxylase, is the next step in the Cahill cycle. The metabolic pathway encounters a physical barrier here since the necessary enzyme is localised in the mitochondrial matrix. The mitochondrial pyruvate carrier (MPC) complex, consisting of dimerised subunits MPC-1 and MPC-2, translocates pyruvate into the mitochondria.
Expression of MPC is under hormonal regulation; increase in circulating pancreatic glucagon during fasting upregulates expression of MPC on mitochondrial outer membranes. Glucagon binds to G protein-coupled receptors and activates adenylate cyclase which increases cAMP production. cAMP phosphorylates PKA which in turn activates the cAMP responsive element binding protein (CREB). CREB, a transcription factor, directly stimulates gluconeogenesis by binding the promoter of the MPC1 and MPC2 genes as well as the promoters of important enzymes involved later in the glucose-alanine cycle. This long-term regulation promotes gluconeogenesis during extended periods of fasting in which blood glucagon concentration is consistently high.
Once in the mitochondria, pyruvate is converted to oxaloacetate by pyruvate carboxylase. The enzyme requires a covalently-bonded prosthetic group, biotin, to act as a carrier of activated CO2. Pyruvate carboxylase consists of four identical subunits each made of four domains: a biotin carboxylase (BC) domain, a biotin carboxyl carrier protein (BCCP), a pyruvate carboxylase (PC) domain, and a PT domain which stabilises the tetramer
First, carbonic acid reacts with ATP to produce activated CO2. This CO2 is then transferred to the biotin attached to the BCCP subunit, releasing a orthophosphate molecule. Finally, the CO2 is transferred to a pyruvate molecule (catalysed in the active site of the PC subunit) to form oxaloacetate.
This is the first irreversible, committed step of the gluconeogenesis pathway and, hence, is a key step for regulation. The energy charge of the cell plays a crucial role in activating pyruvate carboxylase – biotin cannot be carboxylated unless the allosteric regulator acetyl CoA is bound to the enzyme. If the energy charge in the liver cell is low, acetyl CoA concentration will be low and pyruvate will instead be decarboxylated to enter the TCA cycle, inhibiting gluconeogenesis. If acetyl CoA concentration is high, phosphate dehydrogenase (a glycolytic enzyme) is inhibited and pyruvate carboxylase activity is stimulated.
OXALOACETATE –> PHOSPHOENOLPYRUVATE (PEP)
The enzyme that catalyses the conversion of oxaloacetate to PEP is only present in the cytoplasm so the tricarboxylic acid must first be transported out of the mitochondria. The inner mitochondrial membrane is impermeable to oxaloacetate but not to malate; reduction to malate via malate dehydrogenase allows the molecule to be transported by the malate- α-ketoglutarate transporter. In the cytoplasm, malate is then oxidised by cytosolic malate dehydrogenase to reform oxaloacetate.
The malate shuttle serves another important purpose as it enables the transport of reducing equivalents across the outer mitochondrial membrane (which is impermeable to NADH). When malate is converted back to oxaloacetate, cytoplasmic NADH is formed which reduces substrates later in the gluconeogenic pathway.
Oxaloacetate is simultaneously decarboxylated and phosphorylated (using GTP) by phosphoenolpyruvate carboxykinase (PEPCK) to produce PEP. Metal ions, Mn2+ and Mg2+ act as cofactors and stabilise the several negative charges interacting in this reaction.
Cytosolic PEPCK gene expression is hormonally regulated by glucagon via cAMP (as described earlier) as well as by cortisol. This hormone diffuses directly through the phospholipid bilayer of hepatic cells and binds to the glucocorticoid receptor (GR) to form a cortisol-GR complex. The complex then translocates into the nucleus and binds the Glucocorticoid Response Element upstream of the PEPCK gene – activating it. Insulin, on the other hand, downregulates PEPCK expression – inhibiting gluconeogenesis.
By carboxylating and subsequently decarboxylating pyruvate, the phosphorylation of the substrate (typically a very endergonic reaction) is made more favourable. Direct addition of a phosphoryl group has a high ΔG ̊of +31 kJmol-1, making the reverse dephosphorylation reaction irreversible. Decarboxylation drives the gluconeogenic pathway forwards by bypassing this unfavourable step with a much lower ΔG ̊of +0.8 kJmol-1.
PEP à FRUCTOSE-1,6-BISPHOSPHATE
The six following reactions are an exact reversal of the glycolytic pathway and are catalysed by glycolysis enzymes; this is possible as all these reactions are reversible and near equilibrium in hepatic cells – if the metabolite concentrations favour gluconeogenesis, PEP will be converted to fructose-1,6-bisphosphate via:
Phosphoenolpyruvate + H2O ⇌ 2-phosphoglycerate
2-Phosphoglycerate ⇌ 3-phosphoglycerate
3-Phosphoglycerate + ATP ⇌ 1,3-bisphosphoglycerate + ADP
The dephosphorylation of F16BP to F6P, mediated by fructose-1,6-phosphatase (FBPase) and Mg2+ ion cofactors, bypasses another irreversible step of the glycolytic pathway and is a key point of regulation in the gluconeogenic pathway. FBPase is a tetrameric enzyme with an AMP binding site as well as an active site; fructose-2,6-phosphate competitively inhibits FBPase by binding the active site and sterically inhibiting the substrate F16BP from binding whereas AMP inhibits FBPase by binding an allosteric site.
F26BP is an allosteric effector molecule that is not an intermediate of either pathway but regulates both glycolysis and gluconeogenesis. Intracellular concentration of F26BP is under hormonal control; glucagon decreases the concentration whereas insulin increases it. Glucagon stimulates cAMP which activates PKA which phosphorylates a serine residue in the active site of the bi-enzyme PFK-2/FBPase-2. Phosphorylation increases the phosphatase activity of the enzyme, converting F26BP to F6P, so the effector is unable to bind and inhibit FBPase – stimulating gluconeogenesis. Insulin, on the other hand, binds to specific membrane receptors and activates phosphoprotein phosphatase 2A (PP2A) which catalyses the dephosphorylation of PFK-2/FBPase-2. This allows the bi-enzyme to act as a kinase and increase F26BP concentration – inhibiting gluconeogenesis.
AMP acts as a non-competitive inhibitor; upon AMP ligation, hydrogen bonds holding the FBPase tetramer together are disrupted causing a 15 ̊ to 17 ̊ rotation of the upper dimer relative to the lower dimer which converts the enzyme from the R state to the inactive T state.
FBPase regulation plays a crucial role in survival for obligate hibernators. During hibernation, glycolysis must be prioritised so hepatic gluconeogenesis is inhibited. Respiration rate dramatically decreases, creating relatively anoxic conditions in liver tissue which inhibits FBPase by making it more sensitive to allosteric inhibitors (ex. AMP, ADP, Pi) and F26BP. FBPase affinity for its substrate also decreases through covalent modification; for instance, hibernating bats show a 75% decrease in KM compared to euthermic bats.
GLUCOSE-6-PHOSPHATE –> GLUCOSE
Fructose-6-phosphate is readily converted to glucose-6-phosphate by the same glycolytic enzyme that catalyses the reverse reaction since the reaction is maintained close to equilibrium.
Standard gluconeogenesis terminate here in most tissues since glucose-6-phosphate is unable to readily diffuse out of cells and can be converted to glycogen for faster mobilisation during aerobic respiration. However, the interorgan glucose-alanine cycle is unique in its production of free glucose which is transported out of the liver cells and recirculated to the skeletal muscle. This dephosphorylation requires a third bypass of an irreversible step of the glycolytic pathway; glucose-6-phosphatase, a protein complex embedded in the ER, catalyses the reaction instead of hexokinase/glucokinase. Only tissues that maintain blood-glucose homeostasis, such as the liver and kidney, contain these G6Pase complexes.
SUMMARY
The overall reaction for the conversion of alanine to glucose is:
2 Alanine + 10 ATP + CO2⇌ Glucose + urea + 10 ADP + 10 Pi
The alanine-glucose Cahill cycle repurposes carbon skeletons of the liberated amino acid and provides a means of safely exporting and processing amino groups (via the linked Urea cycle) whilst ensuring that the liver bears the energetic burden of gluconeogenesis instead of actively respiring skeletal muscle tissue.
The three main points of regulation in gluconeogenesis:
pyruvate –> oxaloacetate
oxaloacetate –> PEP
F16BP –> F6P
correspond with irreversible steps in the glycolytic pathway. The main forms of regulation are hormonal control via antagonistic glucagon/cortisol and insulin as well as allosteric regulation by acetyl CoA and AMP.
BIBLIOGRAPHY
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Curiosity Killed The Cation 