Membrane-anchored intracellular insulin receptor or insulin-like growth factor-1 receptor elicits ligand-independent downstream signaling.

Neurodegenerative diseases including glaucoma affect insulin signaling, and insulin treatment has been shown to reverse the neurodegenerative loss of dendritic complexity in retinal ganglion cells. Therefore, strategies for enhancing or maintaining insulin signaling are worth pursuing to establish new therapies for these diseases. In the present study, we generated constitutively active insulin receptor (F-iIR) and insulin-like growth factor-1 receptor (F-iIGF1R) using a system that forces membrane localization of the intracellular domains of these receptors by farnesylation. Immunohistochemistry and Western blot analysis revealed that F-iIR and F-iIGF1R caused the activation of ERK and AKT in the absence of ligands in vitro. Our results suggest that in vivo effects of F-iIR and F-iIGF1R on the progression of neurodegenerative diseases should be investigated in the future.

Updates on protein-prenylation and associated inherited retinopathies.

Membrane-anchored proteins play critical roles in cell signaling, cellular architecture, and membrane biology. Hydrophilic proteins are post-translationally modified by a diverse range of lipid molecules such as phospholipids, glycosylphosphatidylinositol, and isoprenes, which allows their partition and anchorage to the cell membrane. In this review article, we discuss the biochemical basis of isoprenoid synthesis, the mechanisms of isoprene conjugation to proteins, and the functions of prenylated proteins in the neural retina. Recent discovery of novel prenyltransferases, prenylated protein chaperones, non-canonical prenylation-target motifs, and reversible prenylation is expected to increase the number of inherited systemic and blinding diseases with aberrant protein prenylation. Recent important investigations have also demonstrated the role of several unexpected regulators (such as protein charge, sequence/protein-chaperone interaction, light exposure history) in the photoreceptor trafficking of prenylated proteins. Technical advances in the investigation of the prenylated proteome and its application in vision research are discussed. Clinical updates and technical insights into known and putative prenylation-associated retinopathies are provided herein. Characterization of non-canonical prenylation mechanisms in the retina and retina-specific prenylated proteome is fundamental to the understanding of the pathogenesis of protein prenylation-associated inherited blinding disorders.

Evaluating protein prenylation of human and viral CaaX sequences using a humanized yeast system.

Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Genetic and pharmacological modulation of lamin A farnesylation determines its function and turnover.

Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.

Physiological adventures in Candida albicans: farnesol and ubiquinones.

SUMMARYFarnesol was first identified as a quorum-sensing molecule, which blocked the yeast to hyphal transition in Candida albicans, 22 years ago. However, its interactions with Candida biology are surprisingly complex. Exogenous (secreted or supplied) farnesol can also act as a virulence factor during pathogenesis and as a fungicidal agent triggering apoptosis in other competing fungi. Farnesol synthesis is turned off both during anaerobic growth and in opaque cells. Distinctly different cellular responses are observed as exogenous farnesol levels are increased from 0.1 to 100 µM. Reported changes include altered morphology, stress response, pathogenicity, antibiotic sensitivity/resistance, and even cell lysis. Throughout, there has been a dearth of mechanisms associated with these observations, in part due to the absence of accurate measurement of intracellular farnesol levels (Fi). This obstacle has recently been overcome, and the above phenomena can now be viewed in terms of changing Fi levels and the percentage of farnesol secreted. Critically, two aspects of isoprenoid metabolism present in higher organisms are absent in C. albicans and likely in other yeasts. These are pathways for farnesol salvage (converting farnesol to farnesyl pyrophosphate) and farnesylcysteine cleavage, a necessary step in the turnover of farnesylated proteins. Together, these developments suggest a unifying model, whereby high, threshold levels of Fi regulate which target proteins are farnesylated or the extent to which they are farnesylated. Thus, we suggest that the diversity of cellular responses to farnesol reflects the diversity of the proteins that are or are not farnesylated.

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences.

The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the a-factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined.

Glutamylation of Npm2 and Nap1 acidic disordered regions increases DNA charge mimicry to enhance chaperone efficiency.

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that, to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

A plant protein farnesylation system in prokaryotic cells reveals Arabidopsis AtJ3 produced and farnesylated in E. coli maintains its function of protecting proteins from heat inactivation.

BACKGROUND: Protein farnesylation involves the addition of a 15-carbon polyunsaturated farnesyl group to proteins whose C-terminus ends with a CaaX motif. This post-translational protein modification is catalyzed by a heterodimeric protein, i.e., farnesyltransferase (PFT), which is composed of an α and a ß subunit. Protein farnesylation in plants is of great interest because of its important roles in the regulation of plant development, responses to environmental stresses, and defense against pathogens. The methods traditionally used to verify whether a protein is farnesylated often require a specific antibody and involve isotope labeling, a tedious and time-consuming process that poses hazardous risks. RESULTS: Since protein farnesylation does not occur in prokaryotic cells, we co-expressed a known PFT substrate (i.e., AtJ3) and both the α and ß subunits of Arabidopsis PFT in E. coli in this study. Farnesylation of AtJ3 was detected using electrophoretic mobility using SDS-PAGE and confirmed using mass spectrometry. AtJ3 is a member of the heat shock protein 40 family and interacts with Arabidopsis HSP70 to protect plant proteins from heat-stress-induced denaturation. A luciferase-based protein denaturation assay demonstrated that farnesylated AtJ3 isolated from E. coli maintained this ability. Interestingly, farnesylated AtJ3 interacted with E. coli HSP70 as well and enhanced the thermotolerance of E. coli. Meanwhile, AtFP3, another known PFT substrate, was farnesylated when co-expressed with AtPFTα and AtPFTß in E. coli. Moreover, using the same strategy to co-express rice PFT α and ß subunit and a potential PFT target, it was confirmed that OsDjA4, a homolog of AtJ3, was farnesylated. CONCLUSION: We developed a protein farnesylation system for E. coli and demonstrated its applicability and practicality in producing functional farnesylated proteins from both mono- and dicotyledonous plants.

Revisiting the truncated lamin A produced by a commonly used strain of Lmna knockout mice.

The Lmna knockout mouse (Lmna-/-) created by Sullivan and coworkers in 1999 has been widely used to examine lamin A/C function. The knockout allele contains a deletion of Lmna intron 7-exon 11 sequences and was reported to be a null allele. Later, Jahn and coworkers discovered that the mutant allele produces a 54-kDa truncated lamin A and identified, by RT-PCR, a Lmna cDNA containing exon 1-7 + exon 12 sequences. Because exon 12 encodes prelamin A's CaaX motif, the mutant lamin A is assumed to be farnesylated. In the current study, we found that the truncated lamin A in Lmna-/- mouse embryonic fibroblasts (MEFs) was predominantly nucleoplasmic rather than at the nuclear rim, leading us to hypothesize that it was not farnesylated. Our study revealed that the most abundant Lmna transcripts in Lmna-/- MEFs contain exon 1-7 but not exon 12 sequences. Exon 1-7 + exon 12 transcripts were detectable by PCR but in trace amounts. We suspect that these findings explain the nucleoplasmic distribution of the truncated lamin A in Lmna-/- MEFs, and subsequent cell transduction experiments support this suspicion. A truncated lamin A containing exon 1-7 sequence was nucleoplasmic, whereas a lamin A containing exon 1-7 + exon 12 sequences was located along the nuclear rim. Our study explains the nucleoplasmic targeting of truncated lamin A in Lmna-/- MEFs and adds to our understanding of a commonly used strain of Lmna-/- mice.

A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine-aliphatic1-aliphatic2-variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.

Quantification of Farnesylated Progerin in Hutchinson-Gilford Progeria Patient Cells by Mass Spectrometry.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal disorder characterized by premature aging and death at a median age of 14.5 years. The most common cause of HGPS (affecting circa 90% of patients) is a de novo heterozygous synonymous single-base substitution (c.1824C>T; p.G608G) in the LMNA gene that results in the accumulation of progerin, an aberrant form of lamin A that, unlike mature lamin A, remains permanently farnesylated. The ratio of progerin to mature lamin A correlates with disease severity in HGPS patients, and can be used to assess the effectiveness of therapies aimed at lessening aberrant splicing or progerin farnesylation. We recently showed that the endogenous content of lamin A and progerin can be measured by mass spectrometry (MS), providing an alternative to immunological methods, which lack the necessary specificity and quantitative accuracy. Here, we present the first non-immunological method that reliably quantifies the levels of wild-type lamin A and farnesylated progerin in cells from HGPS patients. This method, which is based on a targeted MS approach and the use of isotope-labeled internal standards, could be applied in ongoing clinical trials evaluating the efficacy of drugs that inhibit progerin farnesylation.

Regulation of the SNARE protein Ykt6 function by diprenylation and phosphorylation.

For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.

Protein Farnesylation on Nasopharyngeal Carcinoma, Molecular Background and Its Potential as a Therapeutic Target.

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies. NPC is highly metastatic compared to other head and neck carcinomas, and evidence has shown that the metastatic features of NPC are involved in EBV infection. The prognosis of advanced cases, especially those with distant metastasis, is still poor despite advancements in molecular research and its application to clinical settings. Thus, further advancement in basic and clinical research that may lead to novel therapeutic modalities is needed. Farnesylation is a lipid modification in the C-terminus of proteins. It enables proteins to attach to the lipid bilayer structure of cellular membranes. Farnesylation was initially identified as a key process of membrane association and activation of the RAS oncoprotein. Farnesylation is thus expected to be an ideal therapeutic target in anti-RAS therapy. Additionally, more and more molecular evidence has been reported, showing that proteins other than RAS are also farnesylated and have significant roles in cancer progression. However, although several clinical trials have been conducted in cancers with high rates of ras gene mutation, such as pancreatic carcinomas, the results were less favorable than anticipated. In contrast, favorable outcomes were reported in the results of a phase II trial on head and neck carcinoma. In this review, we provide an overview of the molecular pathogenesis of NPC in terms of the process of farnesylation and discuss the potential of anti-farnesylation therapy in the treatment of NPC.

Temperature-Responsive Nano-Biomaterials from Genetically Encoded Farnesylated Disordered Proteins.

Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins' sequence-structure-function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites.

During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum, and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group. In other organisms, farnesylation of Hsp40 orthologs controls their localization and function in resisting environmental stress. In this work, we find that plastidial isopentenyl pyrophosphate (IPP) synthesis and protein farnesylation are required for malaria parasite survival after cold and heat shock. Furthermore, loss of HSP40 farnesylation alters its membrane attachment and interaction with proteins in essential pathways in the parasite. Together, this work reveals that farnesylation is essential for parasite survival during temperature stress. Farnesylation of HSP40 may promote thermotolerance by guiding distinct chaperone-client protein interactions.

Lamin post-translational modifications: emerging toggles of nuclear organization and function.

Nuclear lamins are ancient type V intermediate filaments with diverse functions that include maintaining nuclear shape, mechanosignaling, tethering and stabilizing chromatin, regulating gene expression, and contributing to cell cycle progression. Despite these numerous roles, an outstanding question has been how lamins are regulated. Accumulating work indicates that a range of lamin post-translational modifications (PTMs) control their functions both in homeostatic cells and in disease states such as progeria, muscular dystrophy, and viral infection. Here, we review the current knowledge of the diverse types of PTMs that regulate lamins in a site-specific manner. We highlight methods that can be used to characterize lamin PTMs whose functions are currently unknown and provide a perspective on the future of the lamin PTM field.