Daria Camozzi

Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50-pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

Pre‐Lamin A processing is linked to heterochromatin organization

Pre‐lamin A undergoes subsequent steps of post‐translational modification at its C‐terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre‐lamin A processing in nuclei, induced by expression of mutated pre‐lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non‐farnesylated) pre‐lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl‐K9‐histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre‐lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre‐lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins. J. Cell. Biochem. 102: 1149–1159, 2007.

Diverse lamin-dependent mechanisms interact to control chromatin dynamics Focus on laminopathies

Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals. Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.

Prelamin A is involved in early steps of muscle differentiation

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2alpha were observed. Furthermore, prelamin A was found in a complex with LAP2alpha in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2alpha and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation.

Emerin—prelamin A interplay in human fibroblasts

Background information. Emerin is a nuclear envelope protein that contributes to nuclear architecture, chromatin structure, and gene expression through its interaction with various nuclear proteins. In particular, emerin is molecularly connected with the nuclear lamina, a protein meshwork composed of lamins and lamin‐binding proteins underlying the inner nuclear membrane. Among nuclear lamina components, lamin A is a major emerin partner. Lamin A, encoded by the LMNA gene (lamin A/C gene), is produced as a precursor protein (prelamin A) that is post‐transcriptionally modified at its C‐terminal region where the CaaX motif triggers a sequence of modifications, including farnesylation, carboxymethylation, and proteolytic cleavage by ZMPSTE 24 (zinc metalloproteinase Ste24) metalloproteinase. Impairment of the lamin A maturation pathway causing lamin A precursor accumulation is linked to the development of rare diseases such as familial partial lipodystrophy, MADA (mandibuloacral dysplasia), the Werner syndrome, Hutchinson—Gilford progeria syndrome and RD (restrictive dermopathy).

Prelamin A processing and functional effects in restrictive dermopathy

Laminopathies are an heterogeneous group of human disorders caused by mutations in the lamin A/C gene or in genes coding for lamin-binding proteins. They include several tissue-specific disorders (e.g., Emery-Dreifuss muscular distrophy and dilated cardiomyopathy with conduction defects) or systemic forms including the most severe phenotypes: Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD).1 RD is a lethal neonatal laminopathy causing bone resorption of clavicles, tight translucent skin, anomalous facial features and arthrogryposis.2 RD is a secondary laminopathy, since it is associated with mutations of a lamin-binding protein, the endoprotease ZMPSTE24 involved in lamin A precursor (prelamin A) post-translational processing.3 A common mutation in the ZMPSTE24 gene (c.1085_1086InsT) has been characterized in most RD patients leading to impaired protein expression and prelamin A accumulation in cells. Prelamin A processing consists of four steps: farnesylation of the C-terminal CaaX motif by the enzyme protein farnesyl-transferase, cleavage of the last three aminoacids by ZMPSTE24, carboxymethylation of the C-terminal cysteine by Icmt methyl-transferase and proteolytic removal of the last 18 aminoacids by ZMPSTE24. Thus, four processing intermediates are formed during prelamin A maturation.4 Since also the RCE1 endoprotease catalyses the first cleavage step,3 ZMPSTE24 absence should cause accumulation of farnesylated-carboxymethylated prelamin A. Accumulation of different prelamin A forms causes different effects on nuclear organization:5,6 accumulating farnesylated prelamin A causes nuclear enlargement and mis-shapening, along with changes in the localization pattern of heterochromatin-associated proteins HP1 and tri-H3K9,7 while accumulating non-farnesylated prelamin A produces intranuclear prelamin A and heterochromatin clusters, with re-localization of heterochromatin markers, without nuclear enlargement.5 Also, impaired lamin maturation promotes DNA damage accumulation.8,9 We analyzed prelamin A processing in four different RD cell lines bearing the same ZMPSTE24 mutation2 using an antibody (Sc-6214) directed to a C-terminal epitope of prelamin A.10 The experiments were performed at low (5–12) and at high (16–22) culture passage numbers. Low passage RD cells showed high prelamin A levels, but undetectable mature lamin A; prelamin A was localized exclusively at the nuclear rim by both immunofluorescence and colloidal gold-immunoelectron microscopy; western blot analysis confirmed this data (part A). TEM analysis showed severe nuclear morphology defects (enlarged size, envelope invaginations) and heterochromatin disorganization (areas devoid of heterochromatin) in 10–30% of cells, while most of RD nuclei were normally shaped, though presented nuclear envelope duplications and severe heterochromatin loss (part A). Surprisingly, at high passage numbers, labeling by Sc-6214 was reduced in about 90% of nuclei and undetectable in several cells; only highly dysmorphic nuclei showed intense prelamin A staining. Ultrastructural analysis of high passage fibroblasts revealed improved peripheral heterochromatin organization in 50% of the normally shaped nuclei and peripheral heterochromatin clumps in 5% of nuclei. At this stage (p.16–22), western blot analysis revealed prelamin A as a doublet (part B). To characterize prelamin A forms, we used selective antibodies: 1188-1 (directed to the full-length prelamin A-specific C-terminus sequence) detects both non-farnesylated and farnesylated prelamin A, provided that the CSIM terminal sequence is maintained; 1188-2 (directed to the farnesylated prelamin A-specific C-terminus lacking the SIM sequence) detects farnesylated-carboxymethylated prelamin A.4 1188-1 revealed intense fluorescence at low passage number in both normally shaped and dysmorphic nuclei indicating high levels of full-length prelamin A accumulation, while 1188-2 labelled only dysmorphic nuclei, suggesting that farnesylated-carboxymethylated prelamin A was less represented. At high passage numbers, 1188-2 labelled the normally shaped nuclei, while both antibodies revealed intense fluorescence in highly dysmorphic enlarged nuclei (part C). 1188-1 and SC-6214, but not 1188-2, revealed the prelamin A band in western blot analysis at low passages, confirming the small amount of farnesylated-carboxymethylated prelamin A (non-farnesylated, full-length prelamin A-accumulating control was obtained with Mevinolin11). On the contrary, 1188-2 revealed a faint band in very high passage (p.25) cells, which did not accumulate the prelamin A form(s) detectable by 1188-1 or Sc-6214 (part C). We then immunoprecipitated the whole prelamin A exploiting an anti-lamin A/C N-terminus antibody and revealed the immunoprecipitated bands with anti-prelamin A or anti-farnesyl antibodies confirming farnesylation of prelamin A bands (panel D). Searching for functional defects in RD nuclei, we labeled prelamin A and the heterochromatin marker tri-H3K9 or tri-H4K20. At any passage, dysmorphic nuclei accumulating both prelamin A forms, showed altered distribution (i.e., without clusters) or loss (in some nuclei) of tri-H3K9 fluorescence and disorganization of heterochromatic areas (part D); normally shaped nuclei accumulating prelamin A, showed mostly unaffected tri-H3K9, which was only slightly more clustered in a low percentage of nuclei. Analogous alterations were revealed by tri-H4K20 labelling. In conclusion, we detected significant changes in prelamin A levels and post-translational modifications in all cell lines, depending on the passage number. So far, increased prelamin A levels have been observed in progeroid laminopathies, depending on the passage number or on the patients age.7,12 We show that accumulation of full-length, farnesylated protein form prevails in RD fibroblasts, and that a subpopulation of cells accumulates high levels of both full-length and farnesylated-carboxymethylated prelamin A. Finally, we demonstrate that accumulation of both forms is associated with the most severe abnormalities, i.e., nuclear enlargement and mis-shaping and severe chromatin defects. Our data suggest that the first cleavage step in prelamin A post-translational processing is mostly carried out by the ZMPSTE24 endoprotease, which is not efficiently replaced by other enzyme(s) in RD cells. However, our results suggest activation of alternative endoproteolytic processes, probably when high prelamin A levels are reached.

Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

Latency‐associated human cytomegalovirus glycoprotein N genotypes in monocytes from healthy blood donors

BACKGROUND: The β‐herpesvirus human cytomegalovirus (HCMV) infects a variety of cell types and maintains a lifelong relationship with its host by way of a latent infection in circulating monocytes, myeloid precursor cells, and the hematopoietic progenitor population. Viral strain heterogeneity, shown by gene polymorphisms, has been implicated in the majority of HCMV biologic behaviors. HCMV UL73 encodes the polymorphic envelope glycoprotein N (gN), which shows seven genotypes (gN‐1, gN‐2, gN‐3a, gN‐3b, gN‐4a, gN‐4b, and gN‐4c).