A cryptic promoter in the first exon of the SPG4 gene directs the synthesis of the 60-kDa spastin isoform

<p>Abstract</p> <p>Background</p> <p>Mutations in <it>SPG4 </it>cause the most common form of autosomal dominant hereditary spastic paraplegia, a neurodegenerative disease characterized by weakness and spasticity of the lower limbs due to degeneration of the corticospinal tract. <it>SPG4 </it>encodes spastin, a microtubule-severing ATPase belonging to the AAA family. Two isoforms of spastin, 68 and 60 kDa, respectively, are variably abundant in tissues, show different subcellular localizations and interact with distinct molecules. The isoforms arise through alternative initiation of translation from two AUG codons in exon 1; however, it is unclear how regulation of their expression may be achieved.</p> <p>Results</p> <p>We present data that rule out the hypothesis that a cap-independent mechanism may be involved in the translation of the 60-kDa spastin isoform. Instead, we provide evidence for a complex transcriptional regulation of <it>SPG4 </it>that involves both a TATA-less ubiquitous promoter and a cryptic promoter in exon 1. The cryptic promoter covers the 5'-UTR and overlaps with the coding region of the gene. By using promoter-less constructs in various experimental settings, we found that the cryptic promoter is active in HeLa, HEK293 and motoneuronal NSC34 cells but not in SH-SY-5Y neuroblastoma cells. We showed that the cryptic promoter directs the synthesis of a <it>SPG4 </it>transcript that contains a shorter 5'-UTR and translates the 60-kDa spastin isoform selectively. Two polymorphisms (S44L and P45Q), leading to an early onset severe form of hereditary spastic paraplegia when present in heterozygosity with a mutant allele, fall a few nucleotides downstream of the novel transcriptional start site, opening up the possibility that they may exert their modifier effect at the transcriptional level. We provide evidence that at least one of them decreases the activity of the cryptic promoter in luciferase assays.</p> <p>Conclusion</p> <p>We identified a cryptic promoter in exon 1 of the <it>SPG4 </it>gene that selectively drives the expression of the 60-kDa spastin isoform in a tissue-regulated manner. These data may have implications for the understanding of the biology of spastin and the pathogenic basis of hereditary spastic paraplegia.</p

Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1

BACKGROUND: Anosmin-1, the protein implicated in the X-linked Kallmann's syndrome, plays a role in axon outgrowth and branching but also in epithelial morphogenesis. The molecular mechanism of its action is, however, widely unknown. Anosmin-1 is an extracellular protein which contains a cysteine-rich region, a whey acidic protein (WAP) domain homologous to some serine protease inhibitors, and four fibronectin-like type III (FnIII) repeats. Drosophila melanogaster Kal-1 (DmKal-1) has the same protein structure with minor differences, the most important of which is the presence of only two FnIII repeats and a C-terminal region showing a low similarity with the third and the fourth human FnIII repeats. We present a structure-function analysis of the different DmKal-1 domains, including a predicted heparan-sulfate binding site. RESULTS: This study was performed overexpressing wild type DmKal-1 and a series of deletion and point mutation proteins in two different tissues: the cephalopharyngeal skeleton of the embryo and the wing disc. The overexpression of DmKal-1 in the cephalopharyngeal skeleton induced dosage-sensitive structural defects, and we used these phenotypes to perform a structure-function dissection of the protein domains. The reproduction of two deletions found in Kallmann's Syndrome patients determined a complete loss of function, whereas point mutations induced only minor alterations in the activity of the protein. Overexpression of the mutant proteins in the wing disc reveals that the functional relevance of the different DmKal-1 domains is dependent on the extracellular context. CONCLUSION: We suggest that the role played by the various protein domains differs in different extracellular contexts. This might explain why the same mutation analyzed in different tissues or in different cell culture lines often gives opposite phenotypes. These analyses also suggest that the FnIII repeats have a main and specific role, while the WAP domain might have only a modulator role, strictly connected to that of the fibronectins

Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1

m-AAA proteases cleave OPA1 to ensure a balance of long and short OPA1 isoforms, whereas cleavage by OMA1 causes an accumulation of the short OPA1 variants. (See also companion paper from Head et al. in this issue.

Alternative Splicing of Spg7, a Gene Involved in Hereditary Spastic Paraplegia, Encodes a Variant of Paraplegin Targeted to the Endoplasmic Reticulum

BACKGROUND: Hereditary spastic paraplegia defines a group of genetically heterogeneous diseases characterized by weakness and spasticity of the lower limbs owing to retrograde degeneration of corticospinal axons. One autosomal recessive form of the disease is caused by mutation in the SPG7 gene. Paraplegin, the product of SPG7, is a component of the m-AAA protease, a high molecular weight complex that resides in the mitochondrial inner membrane, and performs crucial quality control and biogenesis functions in mitochondria. PRINCIPAL FINDINGS: Here we show the existence in the mouse of a novel isoform of paraplegin, which we name paraplegin-2, encoded by alternative splicing of Spg7 through usage of an alternative first exon. Paraplegin-2 lacks the mitochondrial targeting sequence, and is identical to the mature mitochondrial protein. Remarkably, paraplegin-2 is targeted to the endoplasmic reticulum. We find that paraplegin-2 exposes the catalytic domains to the lumen of the endoplasmic reticulum. Moreover, endogenous paraplegin-2 accumulates in microsomal fractions prepared from mouse brain and retina. Finally, we show that the previously generated mouse model of Spg7-linked hereditary spastic paraplegia is an isoform-specific knock-out, in which mitochondrial paraplegin is specifically ablated, while expression of paraplegin-2 is retained. CONCLUSIONS/SIGNIFICANCE: These data suggest a possible additional role of AAA proteases outside mitochondria and open the question of their implication in neurodegeneration

CLUH regulates mitochondrial metabolism by controlling translation and decay of target mRNAs

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal–neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.Peer reviewe

Whole-Exome Sequencing Identifies Homozygous AFG3L2 Mutations in a Spastic Ataxia-Neuropathy Syndrome Linked to Mitochondrial m-AAA Proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2Y616C gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2Y616C complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other “mitochondrial” features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes