20FA_CH428A-Advanced Biochemistry

This may help you outline your thoughts as you develop your protocol. Keep in mind, protein purification may take multiple steps/procedures!

Lactate, once considered a waste product of glycolysis, has emerged as a critical regulator of cancer development, maintenance, and metastasis. Indeed, tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor outcome. Lactate mediates cancer cell intrinsic effects on metabolism and has additional non–tumor cell autonomous effects that drive tumorigenesis. Tumor cells can metabolize lactate as an energy source and shuttle lactate to neighboring cancer cells, adjacent stroma, and vascular endothelial cells, which induces metabolic reprogramming. Lactate also plays roles in promoting tumor inflammation and in functioning as a signaling molecule that stimulates tumor angiogenesis. Here we review the mechanisms of lactate production and transport and highlight emerging evidence indicating that targeting lactate metabolism is a promising approach for cancer therapeutics.

Lactate dehydrogenase (LDH) interconverts

pyruvate and lactate with concomitant

interconversion of NADHand NAD1. Although crystal

structures of a variety of LDH have previously

been described, a notable absence has been any of

the three known human forms of this glycolytic

enzyme. We have nowdetermined the crystal structures

of two isoforms of human LDH—the M form,

predominantly found in muscle; and the H form,

found mainly in cardiac muscle. Both structures

have been crystallized as ternary complexes in the

presence of the NADH cofactor and oxamate, a

substrate-like inhibitor. Although each of these isoforms

has different kinetic properties, the domain

structure, subunit association, and active-site regions

are indistinguishable between the two structures.

The pKa that governs the KM for pyruvate for

the two isozymes is found to differ by about 0.94 pH

units, consistent with variation in pKa of the activesite

histidine. The close similarity of these crystal

structures suggests the distinctive activity of these

enzyme isoforms is likely to result directly from

variation of charged surface residues peripheral to

the active site, a hypothesis supported by electrostatic

calculations basedoneachstructure.

Juliana struggles to find worthwhile summer plans, while Kirsten opens the idea of doing undergraduate research. Alvin's missions to display the negative effect of extreme amounts of caffeine not surprisingly go horribly wrong (including parkour). Grant Man appears and pushes Juliana to explore what most interests her

Kirsten's summer plans seem to be coming together quickly, while Juliana is still struggling to get started. Oblivious to it all, Alvin takes them on an adventure that ends up getting them thrown out of the Unicorn Café (who knew that was possible?). Grant Man helps Juliana explore her interests through "digging some trial wells."

Kirsten hits a roadblock when she learns all of the literature she needs to review to get her project together. Juliana goes to see a faculty member connected to the project she wants to do, but she chickens out when she gets there. Grant Man encourages her and shows her how to prepare for the meeting. Alvin decides to start a new student group, NUATM -- the Northwestern Alliance for Throwing Muffins.

Alvin and Juliana try to cheer Kirsten up as she stresses about her literature review, but she is not to be consoled. She retreats to the library, creating a book igloo where Grant Man demystifies the lit review process for her. Juliana gives Alvin a potential idea for a summer research project.

While Alvin uploads his parkour video to raise money for his new project idea, Juliana and Kirsten struggle with writing their research grant proposals. Feeling overwhelmed, Grant Man mysteriously appears to both of them, offering advice to simplify the process, getting them back on track, but sending Grant Man running away at the end.

Alvin finally discovers his parkour plans are a disaster and decides to make a last minute run at getting a grant for his throwing muffins game design idea. He enlists Juliana and Kirsten to help as the clock ticks ominously down to the deadline. Grant Man explains to him the importance of the details connected to his project. Will he get it in on time?

Alvin, Juliana, and Kirsten wait anxiously to hear word on their fate from the dreaded Review Committee. Will Juliana get to go to Bolivia? Did Kirsten explain what she will do in her lab clearly enough? Was Alvin able to throw the whole thing together in 48 hours? The grant winners are finally announced, and they learn about their next adventures!