Browsing by Author "Weiss, Michael A."
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Item 12554 Human Sex Determination: SRY Length Regulates Its Cellular Stability And Hence The Robustness Of Testis Differentiation(Oxford University Press, 2024-10-05) Chen, Yen-Shan; Thomson, Ella; Pelosi, Emanuele; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineThe abundance of transcription factors (TFs) mediated by the rates of degradation are subjected to be a robust to an appropriate level. This regulation via the proteasome is largely controlled by the stability of individual proteins and then could determine the direction of a gene-regulatory network. Insight is obtained through studies of bistable genetic circuits mediated by initiating transcription factors. A model is provided by SRY, a Y-encoded TF that initiates testicular differentiation. Known functions in human SRY (204 residues) majorly cluster in its high mobility group (HMG) box whereas the functions of the N- and C-terminal non-box segments are not well characterized. Here, we have used cell-based and mouse transgenic studies to measure the transcriptional threshold of SRY regulating the balance between development and dysgenesis. Our findings demonstrate a threshold length in the C-terminal domain of human SRY that determines the protein’s proteosome-enforced half-life. In a pre-Sertoli cell model, truncation of SRY resulted in the reduction of intracellular concentration and twofold attenuation of the male-specific GRN. Expression of the 1-164 fragment of human SRY in CRISPR-Cas9-edited XX mice failed to drive male differentiation whereas the 1-200 of SRY initiated male GRN development. This study provides insight into the robustness of human SRY and illustrates a powerful strategy to link biochemical properties in cultured cells and in vivo developmental outcomes. Our study reveals a checkpoint in a key TF initiating a sex-specific GRN, functioning as an experimental “control knob” in development. Our approach probes molecular determinants of cell fate and so promises to extend structure-function studies of SRY to the flanking and relatively obscure non-box domains. This result implies the balance between robustness and evolvability in metazoan is a game of numbers of initial transcription factor in the networks.Item Biosynthesis, structure, and folding of the insulin precursor protein(Wiley, 2018-09) Liu, Ming; Weiss, Michael A.; Arunagiri, Anoop; Yong, Jing; Rege, Nischay; Sun, Jinhong; Haataja, Leena; Kaufman, Randal J.; Arvan, Peter; Biochemistry and Molecular Biology, School of MedicineInsulin synthesis in pancreatic β-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.Item Connecting Rodent and Human Pharmacokinetic Models for the Design and Translation of Glucose-Responsive Insulin(American Diabetes Association, 2020-08) Yang, Jing Fan; Gong, Xun; Bakh, Naveed A.; Carr, Kelley; Phillips, Nelson F.B.; Ismail-Beigi, Faramarz; Weiss, Michael A.; Strano, Michael S.; Biochemistry and Molecular Biology, School of MedicineDespite considerable progress, development of glucose-responsive insulins (GRIs) still largely depends on empirical knowledge and tedious experimentation-especially on rodents. To assist the rational design and clinical translation of the therapeutic, we present a Pharmacokinetic Algorithm Mapping GRI Efficacies in Rodents and Humans (PAMERAH) built upon our previous human model. PAMERAH constitutes a framework for predicting the therapeutic efficacy of a GRI candidate from its user-specified mechanism of action, kinetics, and dosage, which we show is accurate when checked against data from experiments and literature. Results from simulated glucose clamps also agree quantitatively with recent GRI publications. We demonstrate that the model can be used to explore the vast number of permutations constituting the GRI parameter space and thereby identify the optimal design ranges that yield desired performance. A design guide aside, PAMERAH more importantly can facilitate GRI's clinical translation by connecting each candidate's efficacies in rats, mice, and humans. The resultant mapping helps to find GRIs that appear promising in rodents but underperform in humans (i.e., false positives). Conversely, it also allows for the discovery of optimal human GRI dynamics not captured by experiments on a rodent population (false negatives). We condense such information onto a "translatability grid" as a straightforward, visual guide for GRI development.Item Diabetes mellitus due to the toxic misfolding of proinsulin variants(Elsevier, 2013) Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDominant mutations in the human insulin gene can lead to pancreatic β-cell dysfunction and diabetes mellitus due to toxic folding of a mutant proinsulin. Analogous to a classical mouse model (the Akita mouse), this monogenic syndrome highlights the susceptibility of human β-cells to endoreticular stress due to protein misfolding and aberrant aggregation. The clinical mutations directly or indirectly perturb native disulfide pairing. Whereas the majority of mutations introduce or remove a cysteine (leading in either case to an unpaired residue), non-cysteine-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the evolution of insulin has been constrained not only by its structure and function, but also by the susceptibility of its single-chain precursor to impaired foldability.Item Distinct states of proinsulin misfolding in MIDY(Springer, 2021-08) Haataja, Leena; Arunagiri, Anoop; Hassan, Anis; Regan, Kaitlin; Tsai, Billy; Dhayalan, Balamurugan; Weiss, Michael A.; Liu, Ming; Arvan, Peter; Biochemistry and Molecular Biology, School of MedicineA precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.Item Evolution of insulin at the edge of foldability and its medical implications(National Academy of Sciences, 2020-11-24) Rege, Nischay K.; Liu, Ming; Yang, Yanwu; Dhayalan, Balamurugan; Wickramasinghe, Nalinda P.; Chen, Yen-Shan; Rahimi, Leili; Guo, Huan; Haataja, Leena; Sun, Jinhong; Ismail-Beigi, Faramarz; Phillips, Nelson B.; Arvan, Peter; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineProteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ∼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge-excluded due to β-cell dysfunction-suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization.Item In Silico Investigation of the Clinical Translatability of Competitive Clearance Glucose-Responsive Insulins(American Chemical Society, 2023-09-18) Yang, Jing Fan; Yang, Sungyun; Gong, Xun; Bakh, Naveed A.; Zhang, Ge; Wang, Allison B.; Cherrington, Alan D.; Weiss, Michael A.; Strano, Michael S.; Biochemistry and Molecular Biology, School of MedicineThe glucose-responsive insulin (GRI) MK-2640 from Merck was a pioneer in its class to enter the clinical stage, having demonstrated promising responsiveness in in vitro and preclinical studies via a novel competitive clearance mechanism (CCM). The smaller pharmacokinetic response in humans motivates the development of new predictive, computational tools that can improve the design of therapeutics such as GRIs. Herein, we develop and use a new computational model, IM3PACT, based on the intersection of human and animal model glucoregulatory systems, to investigate the clinical translatability of CCM GRIs based on existing preclinical and clinical data of MK-2640 and regular human insulin (RHI). Simulated multi-glycemic clamps not only validated the earlier hypothesis of insufficient glucose-responsive clearance capacity in humans but also uncovered an equally important mismatch between the in vivo competitiveness profile and the physiological glycemic range, which was not observed in animals. Removing the inter-species gap increases the glucose-dependent GRI clearance from 13.0% to beyond 20% for humans and up to 33.3% when both factors were corrected. The intrinsic clearance rate, potency, and distribution volume did not apparently compromise the translation. The analysis also confirms a responsive pharmacokinetics local to the liver. By scanning a large design space for CCM GRIs, we found that the mannose receptor physiology in humans remains limiting even for the most optimally designed candidate. Overall, we show that this computational approach is able to extract quantitative and mechanistic information of value from a posteriori analysis of preclinical and clinical data to assist future therapeutic discovery and development.Item Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface(Cold Spring Harbor Laboratory Press, 2021) Chen, Yen-Shan; Racca, Joseph; Amir, Dan; Haas, Elisha; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineMale sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motif’s basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chain’s para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91(±11)% on SRY overexpression and 65(±17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variant’s specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.Item Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone–receptor activation(National Academy of Sciences, 2021) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark A.; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, M. Dodson; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineLigand-dependent conformational switches are ubiquitous in biological macromolecules, from allosteric proteins to RNA riboswitches. Molecular design of artificial switches provides a general strategy to test relationships between macromolecular structure and function. The present study exploited recent structures of complexes between an ancestral signaling protein (insulin) and the ectodomain of its cellular receptor to insert a metabolite-regulated switch into the hormone. Whereas binding of ligands often stabilizes structure, this design envisioned metabolite-dependent “opening” of a closed, inactive insulin conformation. Assessment of hormone-directed receptor autophosphorylation and a downstream signaling cascade in liver-derived cells demonstrated that binding of metabolite (a monosaccharide) enabled hormonal signaling. These results suggest a mechanism-based strategy to design “smart” glucose-responsive analogs to more safely treat insulin-dependent diabetes mellitus.Item New Horizons: Next-Generation Insulin Analogues: Structural Principles and Clinical Goals(The Endocrine Society, 2022) Jarosinski, Mark A.; Chen, Yen-Shan; Varas, Nicolás; Dhayalan, Balamurugan; Chatterjee, Deepak; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDesign of “first-generation” insulin analogues over the past 3 decades has provided pharmaceutical formulations with tailored pharmacokinetic (PK) and pharmacodynamic (PD) properties. Application of a molecular tool kit—integrating protein sequence, chemical modification, and formulation—has thus led to improved prandial and basal formulations for the treatment of diabetes mellitus. Although PK/PD changes were modest in relation to prior formulations of human and animal insulins, significant clinical advantages in efficacy (mean glycemia) and safety (rates of hypoglycemia) were obtained. Continuing innovation is providing further improvements to achieve ultrarapid and ultrabasal analogue formulations in an effort to reduce glycemic variability and optimize time in range. Beyond such PK/PD metrics, next-generation insulin analogues seek to exploit therapeutic mechanisms: glucose-responsive (“smart”) analogues, pathway-specific (“biased”) analogues, and organ-targeted analogues. Smart insulin analogues and delivery systems promise to mitigate hypoglycemic risk, a critical barrier to glycemic control, whereas biased and organ-targeted insulin analogues may better recapitulate physiologic hormonal regulation. In each therapeutic class considerations of cost and stability will affect use and global distribution. This review highlights structural principles underlying next-generation design efforts, their respective biological rationale, and potential clinical applications.