Pedro Carvalho wants to find out how cells get away with being so sloppy. Before newly made proteins can begin their cellular duties, they must fold into a specific shape. But the process is far from efficient; up to 30 percent of the proteins that a cell manufactures end up malformed. Carvalho wants to know how cells cope with this bevy of misfolded proteins, which can be ineffective or even toxic.
A home microscope given to Carvalho in high school sparked his interest in the workings of cells, but he didn’t have much experience with experiments until he finished an undergraduate degree in his native Portugal and began graduate school at the Dana-Farber Cancer Institute in Boston. “That was the first time I went beyond the textbook,” and he found it captivating. “I got addicted to testing hypotheses.”
Taken with the “fantastic pictures” of chromosomes separating that he saw during a stint as a technician in a fruit fly lab, Carvalho decided to study mitosis in his Ph.D. research with cell biologist David Pellman, now an HHMI investigator. His enthusiasm for mitosis and his facility for experiments led to a pair of significant papers on microtubules. These filaments help form the skeleton that supports cells, and they play a role in tugging chromosomes apart during mitosis.
In a 2001 Journal of Cell Biology paper, Carvalho and his colleagues showed that the yeast protein Bik1p helps microtubules hook onto specialized fasteners on the chromosomes, a necessary step for separating the chromosomes. They also discovered that a protein called Kip2 trundles Bik1p to the ends of the microtubules. That work, published in Developmental Cell in 2004, suggested a way cells can control the stability of the microtubules, which begin to break down when the chromosomes separate.
For his postdoctoral research, Carvalho looked for a project that would allow him to continue studying the workings of cells in his favorite model organism: yeast. After meeting cell biologist and HHMI investigator Tom Rapoport of Harvard Medical School at a journal club, he joined Rapoport’s lab to study how cells handle misshapen proteins, which are associated with a range of diseases, from cystic fibrosis to Parkinson’s.
During his time in Rapoport’s lab, Carvalho filled in crucial molecular details about one cellular system responsible for disposing of these proteins, known as the endoplasmic reticulum–associated degradation (ERAD) pathway. Many proteins fold into shape inside the endoplasmic reticulum, a membrane network in the cell. To get rid of misshapen proteins located there, the cell needs to label them for removal. Carvalho and his colleagues revealed in a 2006 Cell paper that different enzymes mark these proteins, depending on whether the distorted section of the protein is inside the endoplasmic reticulum, stuck in its membrane, or jutting into the cytoplasm.
In a 2010 paper in Cell, he clarified what happens next. Once the faulty proteins are identified as junk, the cell transfers them into the cytoplasm, where other proteins can haul them to the cell’s version of a garbage disposal. Carvalho and his colleagues used a technique called photo-crosslinking, which uses ultraviolet light to spur the misfolded protein to attach to nearby molecules. This approach shed light on the mechanism by which misfolded proteins are moved through the endoplasmic reticulum membrane to the cytoplasm, a topic that has been highly contentious. The work suggested that an enzyme called Hrd1p is responsible not only for tagging defective proteins but also for shipping at least some of them into the cytoplasm.
Since 2010, Carvalho has been running his own lab at the Center for Genomic Regulation in Barcelona, Spain, where he hopes to uncover more about the ERAD pathway. He’s particularly interested in getting a model of the pathway working in a test tube by reassembling it from purified components. Doing so would allow him to answer questions such as whether some of the ERAD enzymes form channels in the endoplasmic reticulum membrane that allow the transport of misfolded proteins.
Although his work has revealed some important evidence about one way of handling misfolded proteins, there’s still plenty to do, he says, adding, “ERAD is not the whole story. There are alternative mechanisms to eliminate aberrant, potentially toxic, proteins, and those are even more poorly understood. ” Carvalho’s goal is to reveal all the alternative mechanisms by which misfolded proteins are disposed of, allowing cells to stay healthy.