Understanding Mysterious Class of Rhomboid-like Proteins

Over the last decade, the rhomboid protein superfamily has been uncovered as regulators of diverse membrane-related processes, and yet, almost nothing is known about the molecular mechanism by which they carry out their functions. These membrane-embedded proteins appear in a remarkable number of biological contexts including development, energy balance, sterol regulation, neuronal function, parasitic invasion, bacterial physiology, and protein quality control.

My research program strives to understand the critical and widely conserved functions of proteins belonging to the rhomboid superfamily. Because the rhomboid superfamily underlies many of the most pressing human diseases such as Alzheimer’s, Parkinson’s, immune disorders, cancer, and some infectious diseases, understanding this mysterious class of proteins will reveal new strategies for targeting multiple pathological conditions.

Vibrant green, animated, rotating image of a protein.
Microscopy image showing small, round, multi-colored yeast cells on a black background.  Fluorescent microscopy image showing two large, blue [ebrafish cells on a black background, featuring endoplasmic reticulum staining.Fluorescent microscopy image showing numerous green-colored zebrafish eggs at various stages of development

A Multi-Scale Approach

We employ a multi-scale approach to understand the rhomboid superfamily at the molecular, cellular, and organismal level. We leverage yeast, human cells, and zebrafish as experimental systems for complete characterization of rhomboid proteins at the mechanistic and cellular level, and then scale these studies to the tissue and organismal scale. Concurrently, we are developing a powerful chemical biology platform for generation of small molecule tools as pharmaceutical leads for modulating rhomboid protein function. Ultimately, my research program holds great promise in revealing new fundamental mechanisms which will be harnessed to tackle rhomboid-related diseases.

Research Highlights

We previously discovered that rhomboid Dfm1 is required for the removal of misfolded membrane proteins from the ER, answering a long-standing question in the protein quality control field that has persisted for 3 decades.

Scientific figure showing membrane structure and interactions. Bottom of the image shows a bilayered membrane with Dmf1 represented by blue columns attached via black string-like structure. Also attached is a representation of Cdc48 and SHP also attached to DMf1 via the same black string. Above the membrane image, shows an arrow facing up next to text reading “Retrotranslocation”. Left of the arrow shows a set of red columns facing up as well as crossed, attached by a similar black string-like structure, with 4 round structures annotated “Ub” attached.

We employed genetics, biophysical, biochemical, and computational approaches to decipher sequence regions of Dfm1 that are critical for membrane substrate engagement and retrotranslocation. For the first time, we elucidated how Dfm1 targets misfolded membrane substrates and show that membrane substrate retrotranslocation requires conserved features of the rhomboid superfamily. Intriguingly, Dfm1's lipid thinning function is required to aid in the extraction of membrane substrates and this action can be extended to its human homolog, Derlin-1, which is implicated in a plethora of diseases such as cancer, cystic fibrosis and viral infection.

Nejatfard et al., 2021, Cell Reports

By employing proximity-based proteomics, high-resolution imaging, biochemical, genetics, and lipidomic approaches, we discovered that rhomboid Dfm1 mediates sphingolipid homeostasis.

Scatterplot showing the distribution of sphingolipid levels. X-axis reads “log2(fold change) and Y-axis reads “-log10(p-value)”. Graph shows a gray, butterfly-shaped distribution of points across an asymptote of 0. 5 orange points labeled “LCB2, TSC3,ORM1,ORM2 and LCB1” are located in the upper right quadrant. Legend shows that orange dots indicate ceramide synthesis pathways.

Sphingolipids constitute a major class of lipids where they carry out essential cellular functions in signaling, structural support, apoptosis to name a few. We discovered that Dfm1 controls ER removal and degradation of Orm2, a negative regulator of the sphingolipid biosynthesis pathway. When Dfm1 is dysfunctional, Orm2 builds up in the ER where sphingolipid levels are dysregulated to toxic levels. Perhaps what is more fascinating is that Dfm1’s role in sphingolipid homeostasis is completely separate from its canonical role in membrane protein quality control. This study provides the first evidence that a rhomboid protein is required for regulating the machineries involved in sphingolipid biosynthesis.

Bhaduri and Aguayo et al., 2022, EMBO Journal

How do cells cope with gross accumulation of misfolded membrane proteins?

Three-paneled figure on a green background where each panel shows a membrane under differing conditions. Panel one shows a membrane with crowded proteins and multiple small, round shapes labeled “Ub” coming off of it. This panel is labeled 1. No growth toxicity and aggregation of non-ubiquitinated membrane substrates. Panel two shows the membrane with more spread out proteins and chains of the round shapes labeled “Ub” still attached. Panel 2 is labeled 2. No growth toxicity and accumulation of solubilized ubiquitinated membrane substrates. Finally, panel three shows a membrane with crowded proteins and chains of the round shapes labeled “Ub” still attached. Panel 3 is labeled 3. Growth toxicity and aggregation of ubiquitinated membrane substrates.

By employing RNA sequencing and our rapid genetic screening approach, we discovered that failure to remove misfolded membrane proteins from the ER causes a novel perturbation of cellular homeostasis that does not trigger the canonical ER stress response pathway. In addition, we found that the source of cell toxicity is from aggregation of misfolded membrane proteins. By employing our solubilization assay, we discovered that the yeast rhomboid Dfm1 surprisingly possesses a chaperone function for disaggregating misfolded membrane proteins, a function that is completely separate from its retrotranslocation function. 

Flagg et al., 2021, Molecular Biology of the Cell
Kandel et al., 2022, PLoS Biology