Brady Lab

Research Overview of the Brady Lab

INTRODUCTION

Our independent laboratory at Penn focuses on how dietary metals regulate cellular signaling and metabolism in both health and disease. Our lab investigates metal-protein interactions that drive key biological processes, revealing new mechanisms in cancer and metabolic dysfunction. To broaden the impact of precision oncology, we have co-developed Probe Enabled Activity Reporting (PEAR), a chemical biology platform that maps protein activity in cancer cells and identifies therapeutic targets often missed by genomic analysis.

RESEARCH PROJECTS

Molecular and Cellular Mechanisms of Copper-Dependent Nutrient Signaling and Metabolism 

The driving force behind our long-term research goals has been the hypothesis that protein activity is, in part, dependent on metals, like copper Cu, in order to ensure that cellular processes essential for proliferation are restricted to optimal growth factor and nutrient conditions. As outlined below, our discovery of molecular targets responsible for Cu sensing and the contribution of Cu to cellular adaptive processes engaged during nutrient scarcity reinforces the essential nature of biological systems to convert Cu abundance into cellular adaptations required for cellular and organismal survival. The overarching goal of our ongoing work is to examine the interplay between mechanisms of Cu-sensing necessary for cellular energy homeostasis and evaluating the necessity of Cu for metabolic flexibility under nutrient and oxygen stress. Completion of these studies we will increase our fundamental knowledge of the molecular and cellular features of Cu-dependent enzymes, cellular processes responsive to Cu, and enable therapeutic targeting of Cu-dependent disease vulnerabilities.

We are building on our novel findings by elucidating mechanisms of: i) Cu-controlled autophagy-lysosomal biogenesis and function,  ii) Cu-mediated metabolic flexibility via direct control of glycolytic flux, and iii) interconnectivity between mitochondrial Cu transport and cytosolic nutrient sensing pathways necessary for metabolism.

Project 1: Elucidate the molecular mechanisms and cellular contexts that underlie Cu-controlled autophagy-lysosomal biogenesis and function. 

Vision: Our discoveries that Cu selectively regulates autophagy at the level of the ULK1/2 kinases and the biogenesis and function of lysosomes required for efficient nutrient recycling indicate that Cu abundance may serves as an endogenous rheostat to control cellular adaptive responses to during nutrient deprivation. Collectively, elucidating the molecular mechanisms and cellular contexts that underlie Cu-controlled autophagy-lysosomal biogenesis and function will allows us to gain insight into the intersection of Cu availability and nutrient sensing mechanisms.

Project 2: Elucidate the molecular mechanisms that underlie Cu-mediated metabolic flexibility via direct control of glycolytic flux. 

Vision: The diverse phenotypes associated with alterations in Cu homeostasis suggest that additional Cu-dependent kinases and cellular processes remain to be identified and fully characterized. Our preliminary data demonstrate that the PKM2 isoform of the PKM gene responsible for generating pyruvate within the final rate-limiting step of glycolysis binds to Cu and this association may impact protein confirmation and regulation and be responsible for the contribution of Cu to metabolic rewiring during nutrient and oxygen stress. Examining Cu-mediated PKM2 activity and glycolytic flux will allows us to establish a direct contribution of Cu to metabolism via a dedicated molecular mechanism.  

Project 3: Interrogate the interconnectivity between mitochondrial Cu transport and cytosolic nutrient sensing pathways necessary for metabolism. 

Vision: While our initial studies largely focused on the consequences of cellular Cu deficiency, our preliminary data provides evidence that mitochondrial matrix Cu communicates Cu availability to cytosolic nutrient signaling pathways to maintain metabolic homeostasis. Since mitochondrial Cu deficiency diverges from whole cell Cu deficiency with respect to communication with cytoplasmic nutrient signaling, we will aim to both characterize the nutrient signaling networks that sense mitochondrial Cu availability and examine whether alterations in either cellular or mitochondrial mechanism serve as a means to communicate mitochondrial Cu deficiency. 

Systematic Mapping of the Intersection of Transition Metal Homeostasis and Cellular Metabolism in Mammalian Cells

Transition metal chemistry is vital for life. Unlike organic nutrients and metabolites that can be synthesized de novo or mobilized from intracellular stores in response to deprivation, metals are unique micronutrients necessary for a diversity array of biological processes that can neither be created nor destroyed. As a result, normal physiology relies on proper dietary metal intake by transporters, dedicated molecular machinery to ensure metal selectivity by metalloenzymes within complex biological systems, and strict homeostatic mechanisms to maintain metal levels within an acceptable range. Further, the conventional framework for transitional metal utilization restricts metalloenzymes as proteins with metal cofactors tightly bound to active sites to facilitate structure and catalysis. Despite the universal role of transition metals for all living organisms, and for human health, how cells adapt to fluctuations in metal availability to maintain metal homeostasis and leverage metal abundance to tune biological processes beyond known metalloprotein active site binding events are poorly understood. 

To address key gaps in our knowledge of molecular machinery necessary for metal homeostasis and novel metal-sensing mechanisms engaged during fluctuations in metal levels, we are leveraging our collective expertise in metallobiology, chemical biology, signal transduction, cellular metabolism, organelle biology, and molecular genetics. We aim to systematically map transition metal homeostasis and its intersection with cellular metabolism in mammalian cells. The overarching goal of our proposal is to harness chemoproteomics and functional genomics to i) elucidate the molecular complexity of metal-protein interactions at subcellular resolution, ii) uncover previously unknown cellular machinery necessary for metal homeostasis, and iii) interrogate the functional interplay between cellular metabolism and metal abundance.

Project 1: Spatially map metal occupancy across the proteome using chemoproteomics.

Vision: The dichotomy between metal availability and metal-protein complex affinities (Mn2+ < Fe2+< Co2+< Cu2+> Zn 2+), in which a metal like Cu binds protein ligands more avidly than Fe but is at lower abundance, dictates that complex cellular environments must overcome this trend to ensure that metalloproteins obtain the correct metal cofactor and adventitious metal-protein complexes are limited to avoid toxicity. In addition, metal imaging probes have revealed the existence of weakly bound, labile metal pools that are at dynamic spatial and temporal equilibrium with exchangeable high affinity, static metal pools. In support of the biological relevance of labile metal pools, our lab and others have expanded the traditional dogma of metal-protein interactions beyond high affinity, static metal binding within active sites to include a new paradigm in metal sensing and protein regulation, termed metalloallostery, whereby transient metal binding to a protein allosteric site positively or negatively controls enzyme activity. We are focusing on the development of methodologies to enable monitoring of metal-protein interactions within a native biological system via spatially resolved chemoproteomics and profiling of metal-protein reactivity changes during cellular adaptations to nutrient availability. 

Project 2: Map metabolic genes and small molecule transporters that are involved in cellular metal homeostasis.

Vision: In addition to proteomic based strategies, we are capitalizing on molecular genetics and functional genomeics to discover components of metal homeostasis machinery. We are focusing on performing CRISPR-based genetic screens to identify genes that sensitize or protect against fluctuations in metal abundance that can be coupled to a novel FACS based genetically encoded reporter of metal-driven transcriptional output with the goal of elucidating previously uncharacterized components and regulators of metal homeostasis.  

Unlocking the Chemical Space of Cancer-Associated Perturbations

Current precision oncology largely depends on genetic profiling to guide targeted therapies. However, tumor biology is shaped not only by mutations but also by epigenetic changes, post-translational modifications, and signaling rewiring—factors reflected in the dynamic tumor proteome. Yet, integrating proteomic insights into clinical decision-making remains challenging due to technological limitations.

Our  work focuses on mechanism-based cancer research at the chemistry–cancer biology interface to develop Probe Enabled Activity Reporting (PEAR)—a novel platform for quantitative tumor proteome profiling. By using chemical biology tools in patient-derived models, we aim to visualize and identify reactive protein targets before and after treatment. We hypothesize that changes in the tumor cell reactome reflect functional adaptations to therapy and can reveal new therapeutic vulnerabilities.

Through interconnected discovery modules, PEAR will i) develop methods to visualize reactive protein targets and ii) identify treatment-induced proteomic