Somatostatin Analogs in Fish-hunting Cone Snail Venoms
Cone snails are a large group of marine predators that use complex venoms to capture their prey rapidly. Owing to their stability, chemical diversity, and target selectivity, cone snail toxins (conotoxins) have been developed as biomedical tools, drug leads, and one approved drug. This study reported the discovery that deep-water, fish-hunting cone snails of the Asprella clade use a predation strategy that is different from the rapid and efficient prey capture previously described for cone snails. Asprella snails inject their venom into the fish and then wait for up to three hours before approaching and eating their incapacitated prey. This dramatically different “ambush-and-assess” hunting behavior suggested that Asprellavenoms contain compounds that manipulate the prey’s behavior through unusual mechanisms. A combination of transcriptomics, proteomics, and venom-guided behavioral assays, was used to show that Asprella snails have evolved highly stable toxin mimetics of the vertebrate hormone somatostatin. Notably, one of these toxins, Consomatin Ro1, specifically activates subtypes of the somatostatin receptor implicated in silencing pain. Consistent with these findings, Consomatin Ro1 provides analgesia in two mouse models of pain, and represents a new lead for the development of non-opioid pain therapeutics.
Somatostatin venom analogs evolved by fish-hunting cone snails: From prey capture behavior to identifying drug leads. Ramiro IBL, Bjørn-Yoshimoto WE, Imperial JS, Gajewiak J, Salcedo PF, Watkins M, Taylor D, Resager W, Ueberheide B, Bräuner-Osborne H, Whitby FG, Hill CP, Martin LF, Patwardhan A, Concepcion GP, Olivera BM, Safavi-Hemami H. Sci Adv. 2022 Mar 25;8(12):eabk1410.
Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins.Koch TL, Ramiro IBL, Salcedo PF, Engholm E, Jensen KJ, Chase K, Olivera BM, Bjorn-Yoshimoto W, Safavi-Hemami H. Mol Evol 2022. Apr 10;39(4):msac075.
Malaria Parasites Require a Key Lipid to Make an Organelle
Structure of p97 Unfolding a Substrate
Proteins must fold into specific structures to carry out their diverse functions. When a protein is no longer needed, cells employ specialized unfolding enzymes to facilitate its degradation. One such abundant enzyme is p97. Owing to its central role in maintaining protein balance, p97 has garnered interest as a target for anticancer and antiviral therapies. Additionally, mutations in p97 have been linked to various degenerative diseases. However, the precise mechanism by which p97 unfolds proteins has remained elusive. To shed light on this process, the Shen and Hill labs utilized electron cryomicroscopy (cryo-EM) to capture high-resolution images of p97 actively engaged in protein unfolding. These images were used to reconstruct 3D structures, revealing how p97 unfolds substrates. The structures showed that five copies of p97 encircle the unfolded protein in a spiral staircase arrangement. Meanwhile, a sixth copy of p97 remains detached from the unfolded protein, transitioning between different positions at the ends of the spiral staircase. These findings suggest that p97 unfolds proteins using a "hand-over-hand" mechanism, where the unfolded protein is pulled through the central channel formed by the spiral arrangement. Understanding this unfolding mechanism will facilitate future efforts in developing therapeutics aimed at modulating p97 function.
Active conformation of the p97-p47 unfoldase complex. Xu Y, Han H, Cooney I, Guo Y, Moran NG, Zuniga NR, Price JC, Hill CP, Shen PS. Nat Commun. 2022 May 12;13(1):2640.
A New Insulin Induces a Continuum of Symmetric and Asymmetric Receptor Conformations
Structural Basis for the Action of Loop Diuretic Drugs
Loop diuretics such as bumetanide (marketed as Bumex) are commonly used to treat patients with high blood pressure and fluid retention. These drugs prevent a sodium-potassium-chloride transport protein in the kidney from absorbing ions and water from urine, so they can promote the loss of excessive electrolytes and water. To ‘see’ how diuretics work in atomic detail, Cao and colleagues determined a co-structure with bumetanide caught in the act of inhibiting the transport protein. The structure shows how bumetanide fits into a pocket and prevents the transport protein from changing shape, freezing it in place so that it can no longer escort ions across the membrane barrier. This new structure will now guide the field in developing diuretics that are more effective and have fewer side effects.
Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide.Zhao Y, Roy K, Vidossich P, Cancedda L, De Vivo M, Forbush B, Cao E. Nat Commun. 2022 May 18;13(1):2747.
Identifying Biological Impacts of SARS-CoV-2 Variants of Concern
Identification of a Unique Cancer Cell Susceptibility to Mitochondrial Protein Accumulation
A Protein that Blocks Virus Budding (with the Department of Human Genetics)
To escape cells and spread infection, HIV and other enveloped viruses must wrap themselves in membranes and bud from producer (infected) cells. To complete the budding process, viruses “steal” membrane cutting machinery from the cell (called the ESCRT pathway). This viral dependence upon host factors creates a step at which cells could, in principle, broadly block viral replication. However, such a defense strategy is complicated by the fact that cells also use the ESCRT pathway to perform other critical functions, including the final step of cell division. This collaborative study from the Elde and Sundquist labs showed that multiple different mammals contain duplicated and truncated genes for one of the ESCRT factors. The encoded “retroCHMP3” proteins potently block release of HIV and many other enveloped viruses. Remarkably, however, retroCHMP3 proteins from primates and mice are not highly toxic because although they delay ESCRT-mediated processes, these delays are well tolerated by cells, but are extremely detrimental to HIV and other viruses. This discovery creates the possibility of engineering mice to express retroCHMP3 and test whether they are broadly protected against enveloped viruses, with the long-term goal using retroCHMP3 to learn how to target the ESCRT pathway in new ways to counter viral infections.
RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis. Rheinemann L, Downhour DM, Bredbenner K, Mercenne G, Davenport KA, Schmitt PT, Necessary CR, McCullough J, Schmitt AP, Simon SM, Sundquist WI, Elde NC. Cell. 2021. Oct 14;184(21):5419-5431.e16.
Mitochondrial Adaptations of Malaria Parasites
Malaria parasites are single-celled eukaryotes that evolved under unique selective pressures with unusual metabolic adaptations compared to the human cells they infect. Most eukaryotes, including humans, have a mitochondrial pathway for fatty acid synthesis whose acyl carrier protein (ACP) and associated cofactor biochemically couple acyl-chain synthesis to electron transport chain assembly and iron-sulfur cluster biogenesis. Sigala and coworkers discovered that malaria parasites lack the ability to synthesize fatty acids in mitochondria, yet curiously retain a divergent ACP homolog that is essential for parasite viability. This unusual ACP binds and stabilizes a protein complex required for iron-sulfur cluster biogenesis, despite lacking the cofactor that is required for this association in most eukaryotes. Loss of ACP disables parasite iron-sulfur cluster synthesis, leading to loss of a key protein in the electron transport chain that uses this cofactor, thereby causing parasite death. Malaria parasites have thus evolved to decouple mitochondrial iron-sulfur biogenesis from fatty acid synthesis. This metabolic adaptation is a shared molecular feature with other related human parasites and identifies a pathogen-specific vulnerability suitable for exploration as a therapeutic target.
Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites. Falekun S, Sepulveda J, Jami-Alahmadi Y, Park H, Wohlschlegel J, Sigala PA. eLife. 2021. Oct 6;10:e71636.
A Disease Afflicting One Cellular Organelle Also Impairs Another
Peroxisome Biogenesis Disorders (PBDs) are a set of genetic diseases that are caused by mutations that impair the proper creation and organization of a cellular organelle called the peroxisome. These diseases have a spectrum of symptoms, but can cause devastating developmental and neurological symptoms. Unfortunately, there is currently little that can be done therapeutically to help these patients. Clinical observations made over the past decades have suggested that patients with PBDs not only have defective peroxisomes, but also have signs of defects in another cellular organelle, the mitochondria. Based on this unexpected connection, Rutter and colleagues developed yeast and cultured mammalian cell models of PBDs to try to understand how a primary defect in peroxisomes might induce loss of mitochondrial function. They found that, even when peroxisomal proteins couldn’t be properly sent to peroxisomes, they were still made and ended up accumulating on mitochondria, leading to impaired function. The mitochondrial quality control protein ATAD1 was found to be sufficient to eliminate these proteins from mitochondria and restore mitochondrial function, including in cells from PBD patients. These observations provide additional insight into the source of some of the symptoms of PBDs and can potentially guide strategies to treat those symptoms.
The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder . Nuebel E, Morgan JT, Fogarty S, Winter JM, Lettlova S, Berg JA, Chen YC, Kidwell CU, Maschek JA, Clowers KJ, Argyriou C, Chen L, Wittig I, Cox JE, Roh-Johnson M, Braverman N, Bonkowsky J, Gygi SP, Rutter J. EMBO Rep. 2021 Oct 5;22(10):e51991.
A Cellular Structure that Protects Against Amino Acid Stress
Amino acids are basic building blocks of all life, used for protein synthesis, metabolic fuel, and biosyntheses. Like most metabolites, cellular amino acid levels must be tightly controlled. Inadequate amino acid availability leads to starvation and associated problems, including sarcopenia, loss of fertility, and decreased immunity. Conversely, amino acid surplus is also highly problematic, and has emerged as a hallmark of many age-related diseases, including cancer and diabetes. Despite our widespread knowledge of how cells sense amino acids and adapt to amino acid starvation, we do not yet fully undertand how cells detect and respond to amino acid excess, and how this excess elicits cellular toxicity if unchecked. This study from Hughes and colleagues outlined a new mechanism by which cells protect themselves from the toxic effects of amino acid excess. When faced with high amino acid levels, cells generate a membrane-bound structure from mitochondria, called the mitochondrial-derived compartment (MDC). MDCs are conserved from yeast to humans and they promote intracellular amino acid catabolism. Loss of MDCs, in combination with other systems that regulate amino acid homeostasis, renders cells highly sensitive to perturbations in amino acids supply, and leads to amino acid-induced cell death. These results outline a new mechanism by which cells regulate nutrient metabolism, and advance our understanding of how cells maintain metabolic homeostasis.
Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress. Schuler MH, English, AM, Xiao T, Campbell TJ, Shaw JM, Hughes AL. Molecular Cell. 2021. Sep 16;81(18):3786-3802.e13.
An Organelle that Regulates Cell Division Timing (with the Department of Oncological Sciences)
When cells divide, they must carefully duplicate their genetic information and distribute the genomes equally into each new daughter cell. Errors in this process lead to genetic mistakes that can lead to cancer or other harmful biological consequences. Cells have therefore evolved quality control mechanisms, called “checkpoints”, that catch errors before the cells are completely separated and it is too late to correct these errors. This collaborative study from the Ullman and Sundquist labs showed that when cell division errors are detected, components that help to separate the daughter cells are sequestered away from the division site into previously unknown structures, which they named abscission checkpoint bodies (ACBs). ACBs also sequester other factors that participate in abscission checkpoint maintenance and as well as factors that contribute to gene expression. These results are key to understanding how cells regulate their division; in particular, they provide a new framework for exploring how this process goes awry and contributes to cancer.
Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing. Strohacker LK, Mackay DR, Whitney MA, Couldwell GC, Sundquist WI, Ullman KS. Elife. 2021. Aug 2;10:e63743.
Mitochondrial Pyruvate Import and Cell Fate (With the Department of Internal Medicine)
To supply their energy needs, cells typically choose between utilizing glucose in the cytoplasm (aerobic glycolysis and lactic acid fermentation) or “burning” pyruvate in the mitochondria (mitochondrial carbohydrate oxidation). Although this is arguably the most fundamental metabolic decision that cells must make, it was not clear how cells import pyruvate into mitochondria to fuel ATP production until the Rutter and Thummel labs identified the heterodimeric MPC1/MPC2 complex as the mitochondrial pyruvate carrier. They also identified and explained the severe metabolic defects found in families with MPC1 gene mutations. They and their collaborators subsequently showed that the choice of whether or not to import pyruvate has far-reaching medical implications because stem cells and most cancer cells are glycolytic (the “Warburg Effect”). They have now shown that aberrant cellular pyruvate metabolism is necessary and sufficient to promote cancer initiation and that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure (with Drakos and colleagues). Collectively, these discoveries have revolutionized our understanding of the role of metabolic decisions in determining cell state and fate, with significant impacts on multiple common diseases.
Regulation of tumor initiation by the mitochondrial pyruvate carrier. Bensard CL, Wisidagama DR, Olson KA, Berg JA, Krah NM, Schell JC, Nowinski SM, Fogarty S, Bott AJ, Wei P, Dove KK, Tanner JM, Panic V, Cluntun A, Lettlova S, Earl CS, Namnath DF, Vázquez-Arreguín K, Villanueva CJ, Tantin D, Murtaugh LC, Evason KJ, Ducker GS, Thummel CS, Rutter J. Cell Metab. 2020 Feb;31(2):284-300.
The pyruvate-lactate axis modulates cardiac hypertrophy and heart failure. C luntun AA, Badolia R, Lettlova S, Parnell KM, Shankar TS, Diakos NA, Olson KA, Taleb I, Tatum SM, Berg JA, Cunningham CN, Van Ry T, Bott AJ, Krokidi AT, Fogarty S, Skedros S, Swiatek WI, Yu X, Luo B, Merx S, Navankasattusas S, Cox JE, Ducker GS, Holland WL, McKellar SH, Rutter J, Drakos SG. Cell Metab. 2021 March 2; 33(3):629-648.e10.
And highlighted in scientific and public outlets, including: Nature Reviews Cardiology.
Ensuring the Accuracy of Chromosome Segregation
Accurate segregation of chromosomes during cell division is fundamental to cellular fitness. However, errors in this process occur in the vast majority of tumor cells, and this appears to be a therapeutic vulnerability. Chromosome segregation is mediated by a highly conserved protein complex, the kinetochore, which attaches chromosomes to microtubules to pull the chromosomes apart. To ensure that this process proceeds accurately, cells use tension to “sense” whether correct kinetochore-microtubule attachments have been made, thereby allowing erroneous attachments to be corrected. Roughly analogous to a children’s “finger trap” toy, the kinetochore increases its grip on the microtubule when greater force is applied, and decreases its grip under low force. How mechanical forces are sensed and transmitted by this protein assembly is poorly understood, and Miller and colleagues therefore investigated how the kinetochore factor, Stu2, is recruited to the kinetochore to carry out its novel mechano-sensing functions. Using protein crystallography, they showed at an atomic level how Stu2 interacts with its kinetochore receptor. Complementary studies showed that the Stu2-kinetochore interaction is critical for the accuracy of chromosome segregation, and furthermore, provided tools that will be instrumental in determining the molecular mechanism of this kinetochore mechanosensor.
Structural basis of Stu2 recruitment to yeast kinetochores. Zahm JA, Stewart MG, Carrier JS, Harrison SC, Miller MP. eLife. 2021. Feb 21;10:e65389.
Faster Antimalarial Activity of Doxycycline
Malaria is a pressing global health challenge, and drug resistance by P. falciparum parasites is a major barrier to treatment efforts. Although new drugs are in development, these medicines will take many years to clear safety hurdles. Doxycycline (DOX) is a key antimalarial drug that is largely limited to prophylaxis due to delayed parasite clearance at current clinical dosage. This slow activity has been thought to be a fundamental limitation of DOX and other drugs that target the essential apicoplast organelle of Plasmodium. Sigala and colleagues discovered, however, that DOX can kill P. falciparum on a faster time-scale via a novel apicoplast-specific mechanism of action at slightly higher drug concentrations than are normally used. These doses can be clinically achieved and are well tolerated. These results expand our understanding of the fundamental antiparasitic mechanisms of DOX and suggest repurposing DOX as a faster-acting antimalarial at higher dosing, where the multiple mechanisms of action are also expected to limit parasite resistance.
Doxycycline has distinct apicoplast-specific mechanisms of antimalarial activity. Okada M, Guo P, Nalder SA, Sigala PA. Elife. 2020 Nov 2;9:e60246.
Reconstituting HIV Replication in vitro
Reverse transcription and integration are the signature events of retrovirus replication and are also targets of successful anti-HIV therapies. Reverse transcription creates a double-stranded DNA copy of the positive-sense viral RNA genome, and integration archives that copy within the genome of the infected cell. However, mechanistic studies of these processes remain challenging because they are performed by viral core particles deep within the infected cell cytoplasm and nucleus. To address this limitation, Sundquist and colleagues reconstituted efficient HIV reverse transcription and integration in a cell-free system, and showed that the system responds appropriated to antiviral compounds. They further found that the viral capsid plays an active role in supporting efficient reverse transcription. Thus, the entire core particle, including the outer capsid shell, is the true viral “replication complex”. This cell-free system should enable systematic analyses of viral replication and integration and thereby help eludicate the first half of the viral life cycle.
Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro. Christensen DE, Ganser-Pornillos BK, Johnson JS, Pornillos O, Sundquist WI. Science. 2020 Oct;370(6513):eabc8420.
Novel Human Insulin Analogs Inspired by Cone Snail Venoms
Faster acting human insulins are needed to improve the efficacy of diabetic insulin pumps. Over the past few years, collaborating teams led by Olivera, Safavi-Hemami, Schlegel, Yandell and Chou have made the remarkable discovery that fish-hunting cone snails use fast-acting insulins to inactivate their prey by inducing hypoglycemia. The researchers characterized these toxins, and used the information gained to design a new fast-acting and very stable human insulin analog that can outperform current competitors for faster onset action. Ongoing collaborating efforts between Chou and Hill have led to a cryo-EM structure of insulin receptor bound to a novel human insulin analog, providing a molecular understanding for the extraordinary properties of these analogs.
Specialized insulin is used for chemical warfare by fish-hunting cone snails. Safavi-Hemami H, Gajewiak J, Karanth S, Robinson SD, Ueberheide B, Douglass AD, Schlegel A, Imperial JS, Watkins M, Bandyopadhyay PK, Yandell M, Li Q, Purcell AW, Norton RS, Ellgaard L, Olivera BM. Proc Natl Acad Sci U S A. 2015 Feb 10;112(6):1743-8.
Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor. Ahorukomeye P, Disotuar MM, Gajewiak J, Karanth S, Watkins M, Robinson SD, Flórez Salcedo P, Smith NA, Smith BJ, Schlegel A, Forbes BE, Olivera B, Hung-Chieh Chou D, Safavi-Hemami H. Elife. 2019 pii: e41574.
A structurally minimized yet fully active insulin based on cone-snail venom insulin principles. Xiong X, Menting JG, Disotuar MM, Smith NA, Delaine CA, Ghabash G, Agrawal R, Wang X, He X, Fisher SJ, MacRaild CA, Norton RS, Gajewiak J, Forbes BE, Smith BJ, Safavi-Hemami H, Olivera B, Lawrence MC, Chou DH. Nat Struct Mol Biol. 2020 Jul;27(7):615-624.
HIV Entry Inhibitors
Our NIH P50 CHEETAH Center supports basic research in HIV structural biology and molecular virology, with the long-term goal of identifying effective new strategies for therapies, vaccines and cures. Such medicines are needed to reduce treatment frequencies, treat drug-resistant patients, prevent new infections, and cure individuals who are already infected. Toward that end, Kay and colleagues have pioneered the development of a platform for creating an entirely new and general class of therapeutic inhibitors, called D-peptides. This year, D-peptide inhibitors of HIV entry were shown to protect macaques very effectively against infection in a primate model of HIV. The leading D-peptide entry inhibitor is now moving into phase I clinical trials.
Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent D-peptide HIV entry inhibitor. Nishimura Y, Francis JN, Donau OK, Jesteadt E, Sadjadpour R, Smith AR, Seaman MS, Welch BD, Martin MA, Kay MS. Proc Natl Acad Sci U S A. 2020 Sep;117(36):22436-22442.
Visualizing the SARS-CoV-2 Life Cycle
SARS-CoV-2 has become the defining disease of the current era, and many biological researchers have redirected their focus to understanding and inhibiting the virus. As a result, we have rapidly gained mechanistic insights into how the virus gains access and hijacks human cells. Iwasa and colleagues have used this information to create detailed molecular animations of different stages of the SARS-CoV-2 life cycle that has been released to the research community and the public. The animations are embedded within a custom web-based user interface that allows users to interact with the animation in order to view annotations (such as protein names and citations) and to ask questions or make comments. This annotation functionality, developed in collaboration with Miriah Meyer (UU SCI), is critical for describing the data used to create the visualization, and also to discuss aspects of the life cycle that are not yet well understood. Based on community feedback, Iwasa and colleagues are iteratively revising the animations to reflect the most current understanding of the viral life cycle.
A Potent, Long-lasting HIV Capsid Inhibitor
The development of antiretroviral drugs has provided life-saving treatments for millions of people living with HIV. These drugs can also prevent new infections via pre-exposure prophylaxis (PrEP). However, problems with drug resistance limits the treatment options of some people living with HIV, and suboptimal adherence to daily drug regimens can adversely affect treatment outcomes and lead to new infections. Hence, there is a need for long-acting drugs that can overcome drug resistance by targeting new classes of viral proteins. This paper described the characterization and phase-1 clinical testing of Lenacapavir (GS-6207), a small molecule that disrupts the functions of HIV capsid protein and is long-acting owing to its high potency and its slow clearance from the body and release from the subcutaneous injection site. Single subcutaneous doses of Lenacapavir maintained antiviral drug concentrations for more than 6 months. The study therefore provides clinical validation for therapies that target the HIV capsid protein, and demonstrate the potential of Lenacapavir as a long-acting agent to treat or prevent infection with HIV. Lenacapavir was developed by Gilead Sciences, leveraging studies of HIV capsid structure and function from the Sundquist and Hill laboratories (and others). Lenacapavir has now successfully completed a phase III clinical trial and FDA approval is pending.
Clinical targeting of HIV capsid protein with a long-acting small molecule. Link JO, (Sundquist WI), et al. Nature. (2020) Aug;584(7822):614-618.
A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model. Yant SR,… Sundquist WI, Cihlar T, Link JO (30 authors). Nature Medicine. 2019 Sep;25(9):1377-84.
Structures and Pharmacology of Cation-Chloride Cotransporters
The cation-chloride cotransporters (CCCs) utilize sodium or potassium gradients to move chloride ions into or out of cells. The CCC family consists of three sodium-dependent cotransporters (e.g., NKCC1) and four sodium-independent cotransporters (e.g., KCC1). CCCs perform fundamental roles in trans-epithelial ion movement, cell volume regulation, chloride homeostasis and inhibitory synaptic transmission. Specifically, NKCCs are key transport proteins involved in salt reabsorption in the kidneys, and the molecular targets of the first line anti-hypertensive loop diuretics. KCCs are emerging targets for the treatment of various brain disorders and psychiatric diseases. Cao and colleagues have determined 3D structures of the free NKCC1 and KCC1 proteins, and of KCC1 bound to an inhibitor (VU0463271). These CCC structures have revealed the architectural design principles in this important class of proteins, their mechanisms of ion binding, and the conformational changes associated with ion transport. The inhibitor-bound KCC1 structure also represents a breakthrough in structural pharmacology of the CCC transporters by helping to reveal the inhibitory mechanisms of anti-hypertensive diuretics.
Structure of the human cation-chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy. Yang X, Wang Q, Cao E. Nat Commun. 2020 Feb;11(1):1016.
Mitochondria and Lysosome Dysfunction in Aging
Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and many age-related and metabolic diseases. Despite long-known connections between these organelles, the function(s) of lysosomes required to sustain mitochondrial health has not been clear. Hughes and colleagues addressed this limitation by showing that the lysosome-like vacuole in yeast maintains mitochondrial respiration by sequestering and organizing intracellular amino acids. Defects in vacuole function impair intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Amongst the different amino acids, cysteine appears most toxic for mitochondria and elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation is sufficient to restore mitochondrial health in vacuole-impaired cells and prevent mitochondrial decline during aging. Overall, this work suggests that cysteine toxicity is a major driver of age-related mitochondrial deterioration, and identifies the lysosome as a new cellular target for minimizing amino acid toxicity.
Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis. Hughes CE, Coody TK, Jeong MY, Berg JA, Winge DR, Hughes AL. Cell. 2020 Jan;180(2):296-310.
Mechanisms of Machines that Unfold Proteins
When a cellular protein has done its job or lost its utility, it needs to be removed, recycled or remodeled. These tasks are performed by members of the ubiquitous family of AAA ATPases ( ATPases associated with diverse cellular activities) that convert the energy of ATP hydrolysis into mechanical forces that can unfold protein aggregates, degrade unwanted proteins, and remodel protein complexes. To learn how AAA ATPases unfold proteins, Hill, Shen, Sundquist and colleagues used electron cryomicroscopy (cryo-EM) to determine structures of several different AAA ATPases in complex with their polypeptide substrates. The team found that each enzyme forms a hexameric ring shaped like a lock washer, with the substrate in the central pore. A “hand-over-hand” mechanism unfolds the polypeptide by pulling it through the pore, driven by cycles of ATP hydrolysis. This mechanism seems to be shared by all members of the highly-conserved AAA ATPase unfoldase family.
Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase. Monroe N, Han H, Shen PS, Sundquist WI, Hill CP. Elife. 2017 Apr 5;6. pii: e24487.
The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets. Han H, Monroe N, Sundquist WI, Shen PS, Hill CP. Elife. 2017 Nov 22;6. pii: e31324.
Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases. Han H, Fulcher JM, Dandey VP, Iwasa JH, Sundquist WI, Kay MS, Shen PS, Hill CP. Elife. 2019 Jun 11;8. pii: e44071.
Structure of the Cdc48 segregase in the act of unfolding an authentic substrate. Cooney I, Han H, Stewart MG, Carson RH, Hansen DT, Iwasa JH, Price JC, Hill CP, Shen PS. Science. 2019 Aug 2;365(6452):502-5. And highlighted in scientific and public outlets, including: @theU, ALS News.
Regulating Mitochondrial Capacity
Cells must decide when to expand mitochondrial capacity to accommodate increased energy demands. Rutter, Winge and colleagues have shown that the ancient mitochondrial fatty acid synthesis system has a profound and unexpected regulatory role in driving mitochondrial biogenesis. The team showed that this is because Acyl Carrier Protein 1 (ACP1), the scaffold on which fatty acids are built, also binds and activates a series of proteins required for mitochondrial biogenesis. However, this only happens when ACP1 is acylated. ACP1 acylation requires the cofactor acetyl-CoA, so this system provides an elegant mechanism for sensing and creating new respiratory capacity to meet demand because acetyl-CoA acts as the universal fuel for respiration and is also the substrate for fatty acid synthesis. Thus, eukaryotic cells adjust the level of active electron transport chain complexes to match the level of acetyl-CoA “fuel” available.
The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis. Van Vranken JG, Jeong MY, Wei P, Chen YC, Gygi SP, Winge DR, Rutter J. Elife. 2016 Aug 19;5. pii: e17828.
ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly. Van Vranken JG, Nowinski SM, Clowers KJ, Jeong MY, Ouyang Y, Berg JA, Gygi JP, Gygi SP, Winge DR, Rutter J. Molecular Cell. 2018 Aug 16;71(4):567-80.
Polycystic Disease Channel Structure
The kidney senses and responds to physiological changes, including pH, ionic strength, pressure, and nutrient levels. This is done with a coupled sensor/ion channel complex called the Polycystic Kidney Disease Channel, which comprises two subunits, PKD1 (the primary sensor), and PKD2 (the channel). Autosomal dominant PKD mutations are amongst the most common monogenic disorders and they lead to untreatable end-stage renal failure. To learn how this system works and is adversely affected by PKD mutations, Cao, Shen and colleagues determined a high resolution cryo-EM structure of the PKD2 channel in lipid nanodiscs. This breakthrough accomplishment provided considerable insight into how the PKD2 channel functions. It was also the first time that cryo-EM was used to determine a high-resolution membrane protein structure at the University of Utah. Finally, it was one of the first membrane protein structures determined within a native-like lipid nanodisc environment, a technology Cao helped to pioneer.
TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Gao Y, Cao E, Julius D, Cheng Y. Nature. 2016 Jun 16;534(7607):347-51.
The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs. Shen PS, Yang X, DeCaen PG, Liu X, Bulkley D, Clapham DE, Cao E. Cell. 2016 Oct 20;167(3):763-73.
Hydrophobic pore gates regulate ion permeation in polycystic kidney disease 2 and 2L1 channels. Zheng W, Yang X, Hu R, Cai R, Hofmann L, Wang Z, Hu Q, Liu X, Bulkley D, Yu Y, Tang J, Flockerzi V, Cao Y, Cao E, Chen XZ. Nature Communications. 2018 Jun 13;9(1):2302.
Animating the HIV Life Cycle
Atomically accurate molecular animations provide a unique opportunity for generating and testing new mechanistic hypotheses, and for scientific communication and public outreach. Iwasa is a leader in the creation of sophisticated, dynamic 3D visualizations of biological processes, and in 2018 she completed and released a molecular animation of the entire HIV life cycle ( http://scienceofHIV.org), which was featured at the International Conference on Retroviral and Opportunistic Infections (CROI March 2018). This work is sponsored by the NIH P50 CHEETAH Center hosted by the Department of Biochemistry, and the animation site will be updated as additional data become available, and to include additional animations that illustrate how antiretroviral therapies work, how a cure might be achieved, how different innate immune restriction factors block HIV replication, and the history of HIV research and treatment in Utah.
This project was highlighted in a series of scientific and public outlets, including: Scientific American magazine, Science Magazine, The NIH Director’s blog, CNET, PBS NOVA and was projected at a large scale in the 2019 Illuminate Salt Lake City Festival .
Distinguishing Between Self and Non-self RNA
Organisms must regulate gene expression and also distinguish their own RNA molecules (self) from the RNAs of invading viruses (non-self). Biochemical and structural studies from Bass, Shen, Iwasa and colleagues revealed how Dicer-2, an RNA processing and antiviral defense enzyme, distinguishes self and non-self by differentially processing double-stranded RNA (dsRNA) substrates as dictated by the unique chemistry at their termini. The study also revealed that human Dicer has evolved distinct activities from invertebrate Dicers, paving the way for altering antiviral defense for therapeutic benefits.
Dicer uses distinct modules for recognizing dsRNA termini. Sinha NK, Iwasa J, Shen PS, Bass BL. Science. 2018 Jan;359(6373):329-34.
Cellular Reprograming by a Herpes Virus
Viruses depend on and modulate their host cellular environments to maximize replication. Studies of viruses can therefore reveal both important aspects of host-pathogen interactions and fundamental cell biology. Viruses often modulate host pathways using proteins, but can also express non-coding RNAs whose functions and mechanisms are largely unknown. Cazalla and colleagues studied the small RNAs from H. saimiri, a herpesvirus that establishes latency in the T cells of New World primates and has the ability to cause aggressive leukemias and lymphomas in non-natural hosts. They showed these RNAs, called HSURs, modulate host gene expression and inhibit host cell death using a novel mechanism in which the viral RNAs inhibit host mRNAs by tethering them to host miRNAs and associated degradation and translation inhibition machinery. This is a completely novel process, not previously observed in cells, but which promises to lead to a fuller understanding of gene regulation in both infected and uninfected cells.
A viral Sm-class RNA base-pairs with mRNAs and recruits microRNAs to inhibit apoptosis. Gorbea C, Mosbruger T, Cazalla D. Nature. 2017 Oct 12;550(7675):275-9.
This project was also highlighted in a series of scientific and public outlets, including: Science
Metabolic Control of Body Temperature
Cold-induced thermogenesis is an energy-demanding process that protects warm blooded animals against reductions in ambient temperature. Villanueva and colleagues demonstrated that in response to cold, the liver switches metabolism to provide acylcarnitines, which are used as fuel by brown fat. Exogenous L-carnitine also rescues the cold sensitivity seen with aging. Thus, this study uncovered an elegant mechanism whereby white adipose tissue provides long-chain fatty acids for hepatic carnitylation, generating plasma acylcarnitines that are used as a fuel source in peripheral tissues.
Global Analysis of Plasma Lipids Identifies Liver-Derived Acylcarnitines as a Fuel Source for Brown Fat Thermogenesis. Simcox J, Geoghegan G, Maschek JA, Bensard CL, Pasquali M, Miao R, Lee S, Jiang L, Huck I, Kershaw EE, Donato AJ, Apte U, Longo N, Rutter J, Schreiber R, Zechner R, Cox J, Villanueva CJ. Cell Metabolism. 2017 Sep 5;26(3):509-522.
De novo Design of Enveloped Protein Nanocages
Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing synthetic self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Sundquist, King, Belnap and colleagues reported the de novo design and characterization of proteins that direct their own assembly and release from human cells within membrane vesicles. These virus-inspired delivery systems can also transfer biological cargoes between cells, and therefore represent an important first step in the development of new synthetic systems for delivering therapeutic cargoes into diseased target cells.
Designed proteins induce the formation of nanocage-containing extracellular vesicles. Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King NP, Sundquist WI. Nature. 2016 Dec 8;540(7632):292-295.
Gene Editing to Correct Sincle Cell Disease
The CRISPR/Cas9 DNA editing system offers the potential for revolutionary new treatments of genetic diseases. Pioneering work by Carroll set the stage for this revolution by characterizing how cells detect and repair double-stranded DNA breaks. Sickle cell disease is common among African Americans and very widespread in tropical regions of Africa and southern Asia. Carroll and his colleagues used the CRISPR/Cas9 gene editing system and a novel delivery method to correct the sickle mutation in adult human hematopoietic stem/progenitor cells. Continuing work on this approach has since improved the efficiency of correction, and treatment of the first patients in clinical trials began in 2021.
Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.DeWitt MA, Magis W, Bray NL, Wang T, Berman JR, Urbinati F, Heo SJ, Mitros T, Muñoz DP, Boffelli D, Kohn DB, Walters MC, Carroll D, Martin DI, Corn JE. Science Translational Medicine. 2016 Oct 12;8(360):360ra134.