Researchers at the Salk Institute for Biological Studies have discovered how a previously uncharacterized microprotein called PIGBOS plays a key role in mitigating a form of cellular stress, and could represent a new target for diverse human diseases. “This study is exciting because cell stress is important in a number of different diseases, including cancer and neurodegeneration,” said Salk professor Alan Saghatelian, PhD, co-corresponding author of the team’s studies, which are reported in Nature Communications. “By understanding the mechanisms behind these diseases, we think we’ll have a better shot at treating them.” Saghatelian and colleagues described their findings in a paper titled, “Regulation of ER stress response by a mitochondrial microprotein.”
Human proteins are typically about 300 amino acids in length, but over recent years scientists have discovered that the mammalian genome harbors possibly thousands of previously uncharacterized small open reading frames (smORFs), which code for much smaller microproteins that may be just 100 or fewer amino acids in length. To date only a handful of smORFs and microproteins have been characterized, the authors commented.
There are three primary branches of the UPR pathway, each of which is mediated by a different ER protein. “Activation of these proteins during UPR initiates signals at the ER that slow down protein expression, increase protein folding, and upregulate degradation of unfolded proteins. If these steps fail to return the cell to homeostasis and prolong activation of UPR, the cells will undergo apoptosis.”
Researchers had previously identified a gene that could code for PIGBOS, but the cellular location of the microprotein and its function weren’t known. Salk postdoctoral researcher and first author Qian Chu, PhD, initially detected PIGBOS microprotein in mitochondria, which are linked with the UPR. “ … mitochondria play a vital role in crosstalk with ER during UPR by providing energy for protein folding in the ER as well as activating apoptosis if the stress remains unmitigated,” the scientists wrote. “However, the communication between the ER and mitochondria in the context of UPR is elusive.” To try and understand more about a potential role for PIGBOS Chu’s team collaborated with co-corresponding author Manor. Manor’s team has expertise in the use of fluorescent protein tags to locate and uncover the function of proteins in cells. “Only now do we really have the sophisticated tools to probe interactions between proteins and see how they work and how they are regulated,” Manor commented.
There were some challenges to the work. PIGBOS was too small to be studied using the green fluorescent protein (GFP) tag, so the team instead exploited an approach called split GFP, which involved fusing just the small beta strand of GFP to the PIGBOS microprotein. This allowed the investigators to study how PIGBOS interacted with other proteins. They found that the microprotein sits on the outer membrane of the mitochondria, where it interacts with a protein called CLCC1, which is part of the ER. “PIGBOS is like a connection to link mitochondria and ER together,” said Chu. “We hadn’t seen that before in microproteins—and it’s rare in just normal proteins.”
The researchers discovered that PIGBOS communicates with CLCC1 to regulate stress and modulate UPR in the ER. Previous studies in mice had linked lower CLCC1 expression levels with increased UPR. Experiments by the Salk team showed that inducing stress in CLCCI knockdown cells led to increased levels of a known marker for UPR. Cells that were engineered to lack PIGBOS were also more sensitive to the chemical tunicamycin, which is an inducer of ER stress, and to other chemicals that cause UPR activation by different mechanisms. Conversely, cells engineered to overexpress PIGBOS were desensitized to UPR. “Taken together, these results suggested that modulating PIGBOS levels can in turn modulate cellular sensitivity towards ER stress,” the scientists stated.
The scientists had not been expecting to see a role for a mitochondrial protein in the unfolded protein response. “These data identified PIGBOS as a heretofore unknown mitochondrial regulator of UPR, and the only known microprotein linked to the regulation of cell stress or interorganelle signaling,” they commented. Together, these results showed that loss of PIGBOS increases cellular sensitivity to ER stress, which in turn increases apoptosis and links PIGBOS levels to the ability of cells to survive stress.”
The findings may help scientists to develop new therapeutic approaches that can target cell stress. “Given the importance of UPR in biology and disease, future studies on PIGBOS’s role in UPR should afford additional insights and may provide methods for regulating this pathway for therapeutic applications,” the team suggested. Chu added, “Going forward, we might consider how PIGBOS is involved in diseases like cancer. In cancer patients, the ER is more stressed than in a normal person, so ER stress regulation could be a good target.”
The researchers are interested in studying the roles of other mitochondrial proteins in ER stress, and in further exploring how PIGBOS functions in an animal model. They are also working to characterize other microproteins. “Microproteins represent a fledgling field,” Saghatelian acknowledged. “But I think this work has really impacted our understanding of the impact that microproteins can have on biochemistry and cell biology.” Manor added, “PIGBOS represents one of a limited set of microproteins that anyone has gone through the effort to characterize. And lo and behold it actually has a very important role.”