Proteome Birthdating Reveals Age-Selectivity of Protein Ubiquitination.

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways.

Long lifetime and tissue-specific accumulation of lamin A/C in Hutchinson-Gilford progeria syndrome.

LMNA mutations cause laminopathies that afflict the cardiovascular system and include Hutchinson-Gilford progeria syndrome. The origins of tissue specificity in these diseases are unclear as the lamin A/C proteins are broadly expressed. We show that LMNA transcript levels are not predictive of lamin A/C protein levels across tissues and use quantitative proteomics to discover that tissue context and disease mutation each influence lamin A/C protein's lifetime. Lamin A/C's lifetime is an order of magnitude longer in the aorta, heart, and fat, where laminopathy pathology is apparent, than in the liver and intestine, which are spared from the disease. Lamin A/C is especially insoluble in cardiovascular tissues, which may limit degradation and promote protein stability. Progerin is even more long lived than lamin A/C in the cardiovascular system and accumulates there over time. Progerin accumulation is associated with impaired turnover of hundreds of abundant proteins in progeroid tissues. These findings identify impaired lamin A/C protein turnover as a novel feature of laminopathy syndromes.

The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate.

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the effect of spatial position on heterochromatin function has not been defined. Here, we remove three nuclear lamins and lamin B receptor (LBR) in mouse embryonic stem cells (mESCs) and show that heterochromatin detaches from the nuclear periphery. Mutant mESCs sustain naïve pluripotency and maintain H3K9me2 across the genome but cannot repress H3K9me2-marked genes or transposons. Further, mutant cells fail to differentiate into epiblast-like cells (EpiLCs), a transition that requires the expansion of H3K9me2 across the genome. Mutant EpiLCs can silence naïve pluripotency genes and activate epiblast-stage genes. However, H3K9me2 cannot repress markers of alternative fates, including primitive endoderm. We conclude that the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of H3K9me2-marked heterochromatin to shape cell fate decisions.